R2R1 / 2 in diagnosis and treatment

Abstract

To provide compounds and methods for use in the treatment of cardiac and pulmonary diseases as well as cancer.SOLUTION: The present invention provides a polynucleotide comprising the R2R1 / 2 gene, for use in treating one or more diseases and / or conditions selected from the group consisting of: (a) cardiac diseases and / or conditions; (b) pulmonary diseases and / or conditions.; and (c) cancer, wherein the R2R1 / 2 gene comprises a sequence selected from the group consisting of a plurality of specific sequences, or a sequence at least 60% identical thereto.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel genes that sustain the development / maintenance of the fine three-dimensional structure of the lung, as well as pharmaceuticals and compositions for treating pulmonary diseases and / or conditions. [Background technology]

[0002] Expression of intermediate filament genes (Krt6), EDC (Epidermal Differentiation Complex) genes, and SCC (Stratified epithelium-secreted protein gene complex) genes allows cells to survive in hostile environments. This expression profile leads to squamous differentiation, a hallmark of skin epithelial cells. We have found that this transcriptional program is also crucial for the refinement of lung structure (late branching morphogenesis). Cells in the distal airways and blood vessels require an oxygen-rich environment to achieve a flat shape and mechanical flexibility. Proteins encoded by intermediate filaments and the associated EDC and SCC genes enable this type of cell shape and flexibility. At the same time, the lung must maintain progenitor cells capable of differentiating into cells capable of assembling these proteins. These progenitor or basal cells typically express the intermediate filament gene Krt14. The lung is similar to the skin in that it is exposed to a great deal of mechanical and oxidative stress. As mentioned above, the squamous differentiation program is the first line of defense. Cells lining the distal airways and blood vessels of the lung must resist this stress and, in the event of cell death, must be replaced by a pool of progenitor cells.

[0003] Dysfunction of this system results in two main human pathologies. 1. Bronchopulmonary dysplasia (BPD): The lungs of premature newborns are more susceptible to lung injury, including mechanical stress. Similarly, the lungs of premature newborns who survive lung injury often heal with significant scarring or bronchopulmonary dysplasia (BPD). These lungs exhibit poor or inadequate regenerative capacity. 2. Chronic Obstructive Pulmonary Disease (COPD): The lungs of adult COPD patients appear to respond inappropriately to noxious stimuli, such as smoking. These lungs develop a barrier against these stimuli, but in the event of cell death, their lack of regenerative capacity leads to deformity of the lung's airways and blood vessels. Both BPD and COPD result in significant morbidity and mortality. Summary of the Invention [Problem to be solved by the invention]

[0004] Novel genes that sustain the development / maintenance of the fine three-dimensional structure of the lung, as well as pharmaceuticals and compositions for treating pulmonary diseases and / or conditions, are provided. [Means for solving the problem]

[0005] 1)(a) Cardiac disease and / or condition; (b) pulmonary diseases and / or conditions; and (c) Cancer; A pharmaceutical composition for the treatment of one or more diseases and / or conditions selected from the group consisting of R2R 1 / 2 A pharmaceutical composition comprising a nucleic acid encoding a protein, said nucleic acid having a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10 and 11, or a sequence at least 60% identical thereto. 2) (a) cardiac disease and / or condition; (b) pulmonary diseases and / or conditions; and (c) Cancer; A pharmaceutical composition for the treatment of one or more diseases and / or conditions selected from the group consisting of R2R 1 / 2 The R2R protein comprises a polypeptide. 1 / 2The pharmaceutical composition, wherein the protein is encoded by a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 3, 6, 9 and 12, or a sequence at least 60% identical thereto. 3) (a) cardiac disease and / or condition; (b) pulmonary diseases and / or conditions; and (c) cancer; A pharmaceutical composition for the treatment of one or more diseases and / or conditions selected from the group consisting of R2R 1 / 2 The pharmaceutical composition comprising an antisense oligonucleotide capable of regulating gene expression. 4) R2R 1 / 2 An antibody or antigen-binding fragment thereof capable of binding to a protein or a fragment thereof. 5) R2R 1 / 2 4) The antibody according to 4), wherein the protein is encoded by a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 3, 6, 9 and 12, or a sequence at least 60% identical thereto. 6)(a) cardiac disease and / or condition; (b) pulmonary diseases and / or conditions; and (c) cancer; A pharmaceutical composition comprising the antibody according to 4) or 5) for use in treating one or more diseases and / or conditions selected from the group consisting of: 7) comprising oligonucleotide probes and / or primers designed to hybridize under stringent conditions to all or part of a sequence selected from the group consisting of SEQ ID NOs: 1; 2; 4; 5; 7; 8; 10 and 11; (a) cardiac diseases and / or conditions; (d) pulmonary diseases and / or conditions; (c) cancer; and (d) Susceptibility to or predisposition to any of (a) to (c) A kit for use in a method for diagnosing one or more diseases and / or conditions selected from the group consisting of: The method comprises: determining whether the R2R in a sample provided by the subject being tested is 1 and / or R2R 2detecting a level of gene expression in the sample; 1 and / or R2R 2 detection of an abnormality at the gene level is indicative of one or more of the diseases and / or conditions set forth in (a) to (c) above and / or susceptibility or predisposition thereto; The kit. 8) The diagnosis is A kit according to 7), which comprises the use of oligonucleotide probes and / or primers designed to hybridize under stringent conditions to all or part of a sequence selected from the group consisting of SEQ ID NOs: 1; 2; 4; 5; 7; 8; 10 and 11. 9) R2R 1 and / or R2R 2 A method for identifying or producing an agent that modulates the expression of a gene, comprising: 1 and / or R2R 2 The gene is contacted with the test drug, and R2R 1 / 2 The method further comprising detecting modulation of gene expression. 10) A pharmaceutical composition comprising a nucleic acid having the sequence of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10 or 11 or a nucleic acid having a sequence at least 60% identical thereto. 11) R2R having the sequences of SEQ ID NOs: 3, 6, 9 and 12 1 / 2 A pharmaceutical composition comprising the protein or a protein having a sequence at least 60% identical thereto. 12) Animal models for the study of cardiac and / or pulmonary diseases or conditions or cancer, comprising: 1 / 2 Said animal model is created by gene modulation or disruption. The present invention will now be described in more detail with reference to the following drawings: [Brief explanation of the drawings]

[0006] [Figure 1]Comparison of endothelial and epithelial cells from Vegf+ / + at E16.5. Volcanoplot (based on LIMMA) showing the significance of each gene on the y-axis when testing the effect of cell origin. The x-axis shows the fold change when comparing the two tissues. Ker+ cells express prototypic epithelial genes, while Il+ cells transcribe typical endothelial genes. Endothelial Cldn5 and epithelial Foxa1 are highlighted because both were also present as top genes on the y-axis (PC2) of the unsupervised spectral map analysis (see Figure 3). [Figure 2] Schematic diagram of the laser capture microdissection process. Embryonic thorax (A) was cut into 8 mm sections and placed on slides (B). Double immunohistochemical staining of these tissue sections (C) with anti-pan keratin and GS-IB4 isolectin separated lung airway cells with epithelial (D) characteristics from surrounding cells with endothelial (E) characteristics. Specific cell populations isolated by laser capture microdissection are shown in F and G (L = lung, H = heart). [Figure 3]Figure 1 shows a spectral map analysis using the first principal component (PC1 on the x-axis) and the second principal component (PC2 on the y-axis). The time effect, or fetal age, was revealed in PC1 (which could explain 35% of the variance in the dataset). Gene probes for genes expressed in late fetal stages, such as Sftpc, were located farther along the x-axis. Differences in cellular origin (il+ cells vs. ker+ cells) were seen in PC2. Gene probes for epithelial genes ('EPI') were located farther along the y-axis, in contrast to gene probes for the endothelial gene ('ENDO') family. In the spectral map according to PC1 (fetal age) and PC2 (cellular origin), different sample groups were separated. ker+ samples clustered on the epithelial side of the y-axis, and il+ samples clustered on the endothelial side. Samples were also assigned along the x-axis according to their fetal age. Late fetal samples were located farther along the x-axis. The following panels show three expression profiles. (1) those showing differences between epithelial and endothelial cells in Foxa1 expression, (2) those showing an effect of fetal age in Afp expression, and (3) those showing a genotype-dependent profile of Hmr expression with respect to fetal age. [Figure 4]Comparison of epithelial cells from the Vegf+ / + and Vegf120 / 120 genotypes. A. Volcano plot (based on LIMMA) showing the significance of each gene on the y-axis to test whether the expression profiles across embryonic age differ between the Vegf+ / + and Vegf120 / 120 genotypes. The x-axis shows the fold change in induction when comparing the two genotypes, Vegf+ / + vs. Vegf120 / 120, at E16.5. Upregulation of Krt6a, EDC, and SCC genes in wild-type Ker+ cells is highlighted. B. Airway epithelial cells in E16.5 Vegf+ / + lungs shown by anti-cytokeratin 4, 5, 6, 8, 10, 13, and 18 staining. Cells in the distal airways develop a flat or squamous cell morphology (yellow arrows) in contrast to their proximal counterparts (white arrows). C. Differential expression allocation along chromosome 3, highlighting gene downregulation in EDCs of Vegf120 / 120 compared to wild type. [Figure 5]A. IFs (red) anchored to desmosomes containing Dsc1 (orange). Pkp1 (purple) connects intermediate keratin filaments to cadherin proteins at adherens junctions (yellow). Pkp1 also regulates the protein content of desmosomes. EDC and SCC cluster genes interact with intermediate filament keratins. Asterisks highlight upregulated genes and gene clusters in the intermediate filament network. Upregulation of Eps8l1 (encoding an actin filament end-capping protein) coordinates intermediate filaments through actin remodeling. B. Volcano plot (based on LIMMA) showing the significance of each gene on the y-axis to test whether the expression profile across embryonic age differs between endothelial cells with the Vegf+ / + and Vegf120 / 120 genotypes. The x-axis shows the fold change in induction comparing the two genotypes, Vegf+ / + vs. Vegf120 / 120, at E16.5. Krt, Dsc1, Pkp1, EDC, and SCC cluster genes are highlighted. The Krt genes upregulated in wild-type Vegf+ / + il+ cells are distinct from those expressed in comparable wild-type ker+ cells. Krt14 and Krt1 are trademark basal cell genes. C. Immunofluorescence image (40x magnification) of GS-IB4 isolectin-stained cells (il+ cells) in wild-type E16.5 embryonic lung. il+ cells follow the same structural pattern as ker+ cells in the distal airway. [Figure 6]Volcano plot based on LIMMA analysis of anti-cytokeratin 4-5-6-8-10-13-18 stained cells (Linear Models for Microarray Data: Differences in expression profiles across embryonic age between Vegf120 / 120 knockout and wild-type littermates were tested using a linear interaction of Vegf genotype and time). The RIKEN cDNA 2200001I15 gene and the RIKEN cDNA 2310002J15 gene are highlighted. [Figure 7] Volcano plot based on LIMMA analysis of GS-IB4-binding cell populations (linear model for microarray data: differences in expression profiles across embryonic age between Vegf120 / 120 knockout and wild-type littermates were tested using a linear interaction of Vegf genotype and time). The RIKEN cDNA 2200001I15 and RIKEN cDNA 2310002J15 genes are highlighted. [Figure 8] Figure 1 shows the alignment of the cDNA sequences of the human hR4RA transcript (=human R2R1) versus the mouse R4Ra transcript (=mouse R2R1). Note that the largest contig constructed from the sequenced RIKEN cDNA 2200001I15 gene clone is designated 'mouse R4Ra transcript (=mouse R2R1)'. [Figure 9] FIG. 1 shows the protein sequence alignment of translated human hR4RA (=human R2R1) transcripts versus translated mouse R4Ra (=mouse R2R1) transcripts. [Figure 10-1] Figure 1 shows the alignment of the cDNA sequences of the human hR4RD transcript (=human R2R2) versus the mouse R4Rd transcript (=mouse R2R2). Note that the largest contig constructed from the sequenced RIKEN cDNA 2310002J15 gene clone is designated 'mouse R4Rd transcript (=mouse R2R2)'. [Figure 10-2] Figure 10-1 continued. [Figure 10-3]Figure 10-2 continued. [Figure 11] FIG. 1 shows the protein sequence alignment of the translated human hR4RD (=human R2R2) transcript versus the translated mouse R4Rd (=mouse R2R2) transcript. [Figure 12] Figure 1 shows VEGF-A (Vegf164)-dependent upregulation of R2R1 in the ventricular septum during mouse embryonic development. Red = wild type, blue = VEGF120 / 120 knockout mice lacking the Vegf164 isoform. [Figure 13] FIG. 1 shows the time course (24 to 72 hours) of human R2R2 homolog (=C9orf196) expression in human adult primary lung epithelial cells. [Figure 14A] Figure 1 shows that siRNA knockdown of VEGF165 results in knockdown of genes involved in the basal and squamous differentiation programs. siRNA knockdown in human primary bronchial epithelial cells (PBECs) is a reliable model for the effects of gene knockdown of a gene of interest. (a) RTqPCR: siRNA-mediated knockdown of VEGFA and VEGF165 expression results in knockdown of KRT14 (a basal cell marker) expression. The relative expression of KRT14 (normalized to PGK1), VEGFA, and VEGF165 is plotted against siRNA. Two negative controls (designated nr10 and 11) are included. An siRNA directed against KRT14 expression is included as a positive control. siRNA VEGFA is directed against all VEGFA isoforms. [Figure 14B]Figure 1 shows that siRNA knockdown of VEGF165 results in knockdown of genes involved in the basal and squamous differentiation programs. siRNA knockdown in human primary bronchial epithelial cells (PBECs) is a reliable model for the effects of gene knockdown of a gene of interest. (a) RTqPCR: siRNA-mediated knockdown of VEGFA and VEGF165 expression results in knockdown of KRT14 (a basal cell marker) expression. The relative expression of KRT14 (normalized to PGK1), VEGFA, and VEGF165 is plotted against siRNA. Two negative controls (designated nr10 and 11) are included. An siRNA directed against KRT14 expression is included as a positive control. siRNA VEGFA is directed against all VEGFA isoforms. [Figure 14C] Figure 1 shows that siRNA knockdown of VEGF165 results in knockdown of genes involved in the basal and squamous differentiation programs. siRNA knockdown in human primary bronchial epithelial cells (PBECs) is a reliable model for the effects of gene knockdown of a gene of interest. (a) RTqPCR: siRNA-mediated knockdown of VEGFA and VEGF165 expression results in knockdown of KRT14 (a basal cell marker) expression. The relative expression of KRT14 (normalized to PGK1), VEGFA, and VEGF165 is plotted against siRNA. Two negative controls (designated nr10 and 11) are included. An siRNA directed against KRT14 expression is included as a positive control. siRNA VEGFA is directed against all VEGFA isoforms. [Figure 15A]A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15B] A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15C] A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15D]A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15E] A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15F] A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15G]A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15H] A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15I] A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 15J]A-J: siRNA-mediated knockdown of VEGFA in PBECs led to global gene expression changes that exactly match those observed in Vegf120 / 120 knockout mice. Graphs (a)-(j) depict the significance of gene expression changes 24 hours after administration of VEGF-directed siRNA. Graphs (a)-(j) are generated by automated pathway analysis. All pathways are involved in keratinocyte differentiation, basal cell regeneration, and squamous differentiation. [Figure 16A]A-C: siRNAs for human R2R1 and R2R2 homologs. Human R2R1 homolog(s) comprise the FAM25 family, which includes seven human paralogs designated FAM25A, FAM25B, FAM25C, FAM25D, FAM25E, FAM25G, and FAM25HP. These are human homologs of the mouse sequence encoded by the cDNA sequence designated 2200001I15Rik or RIKEN cDNA 2200001I15. The similarity of FAM25 paralogs precludes specific RTqPCR of different paralogs. FAM25 paralogs are also not present on Affymetrix Human Gene expression arrays, such as HT HG-U133 or HT HG-U219. We selected the RTqPCR primer-probe set Hs04194072_m1 (Applied Biosystems) to measure differential gene expression of the FAM25 family. Applied Biosystems states that this primer-probe set does not differentiate between different paralogs. Three siRNAs designed against the FAM25 family were selected for their ability to downregulate Hs04194072_m1 expression in PBECs. We numbered these siRNAs nr18, 20, and 22, respectively. Graph (a) shows the downregulation of FAM25 (Hs04194072_m1) expression (normalized to PGK1) in PBECs 24 hours after administration of each siRNA. Two siRNAs (numbered 15 and 17) designed against C9orf169, a human R2R2 homologue, at concentrations of 5 and 20 nM, respectively, were selected based on their ability to downregulate C9orf169 expression in PBECs. C9orf169 expression was assessed by microarray analysis and RT-qPCR. The probe for C9orf169 is present on the Affymetrix HT HG-U219 Human Gene Expression Array. Graph (b) shows the downregulation of C9orf169 expression (assessed by microarray analysis) in PBECs 24 hours after administration of each siRNA.Administration of siRNA directed against the FAM25 family or VEGFA had no effect on C9orf169 expression. siRNA directed against R2R1 (human FAM25 family) and R2R2 (human C9orf169) downregulated KRT14 expression, a response similar to that observed after administration of siRNA directed against VEGFA and VEGF165. Knockdown of the FAM25 family and C9orf169 by siRNA resulted in downregulation of KRT14 gene expression (a basal cell marker). This response was most pronounced after administration of siRNA directed against C9orf169. Graph (c) shows the downregulation of KRT14 expression (normalized to PGK1) in PBECs 24 hours after administration of each siRNA. For comparison, siRNA directed against VEGFA and KRT14 are also included. [Figure 16B]A-C: siRNAs for human R2R1 and R2R2 homologs. Human R2R1 homolog(s) comprise the FAM25 family, which includes seven human paralogs designated FAM25A, FAM25B, FAM25C, FAM25D, FAM25E, FAM25G, and FAM25HP. These are human homologs of the mouse sequence encoded by the cDNA sequence designated 2200001I15Rik or RIKEN cDNA 2200001I15. The similarity of FAM25 paralogs precludes specific RTqPCR of different paralogs. FAM25 paralogs are also not present on Affymetrix Human Gene expression arrays, such as HT HG-U133 or HT HG-U219. We selected the RTqPCR primer-probe set Hs04194072_m1 (Applied Biosystems) to measure differential gene expression of the FAM25 family. Applied Biosystems states that this primer-probe set does not differentiate between different paralogs. Three siRNAs designed against the FAM25 family were selected for their ability to downregulate Hs04194072_m1 expression in PBECs. We numbered these siRNAs nr18, 20, and 22, respectively. Graph (a) shows the downregulation of FAM25 (Hs04194072_m1) expression (normalized to PGK1) in PBECs 24 hours after administration of each siRNA. Two siRNAs (numbered 15 and 17) designed against C9orf169, a human R2R2 homologue, at concentrations of 5 and 20 nM, respectively, were selected based on their ability to downregulate C9orf169 expression in PBECs. C9orf169 expression was assessed by microarray analysis and RT-qPCR. The probe for C9orf169 is present on the Affymetrix HT HG-U219 Human Gene Expression Array. Graph (b) shows the downregulation of C9orf169 expression (assessed by microarray analysis) in PBECs 24 hours after administration of each siRNA.Administration of siRNA directed against the FAM25 family or VEGFA had no effect on C9orf169 expression. siRNA directed against R2R1 (human FAM25 family) and R2R2 (human C9orf169) downregulated KRT14 expression, a response similar to that observed after administration of siRNA directed against VEGFA and VEGF165. Knockdown of the FAM25 family and C9orf169 by siRNA resulted in downregulation of KRT14 gene expression (a basal cell marker). This response was most pronounced after administration of siRNA directed against C9orf169. Graph (c) shows the downregulation of KRT14 expression (normalized to PGK1) in PBECs 24 hours after administration of each siRNA. For comparison, siRNA directed against VEGFA and KRT14 are also included. [Figure 16C]A-C: siRNAs for human R2R1 and R2R2 homologs. Human R2R1 homolog(s) comprise the FAM25 family, which includes seven human paralogs designated FAM25A, FAM25B, FAM25C, FAM25D, FAM25E, FAM25G, and FAM25HP. These are human homologs of the mouse sequence encoded by the cDNA sequence designated 2200001I15Rik or RIKEN cDNA 2200001I15. The similarity of FAM25 paralogs precludes specific RTqPCR of different paralogs. FAM25 paralogs are also not present on Affymetrix Human Gene expression arrays, such as HT HG-U133 or HT HG-U219. We selected the RTqPCR primer-probe set Hs04194072_m1 (Applied Biosystems) to measure differential gene expression of the FAM25 family. Applied Biosystems states that this primer-probe set does not differentiate between different paralogs. Three siRNAs designed against the FAM25 family were selected for their ability to downregulate Hs04194072_m1 expression in PBECs. We numbered these siRNAs nr18, 20, and 22, respectively. Graph (a) shows the downregulation of FAM25 (Hs04194072_m1) expression (normalized to PGK1) in PBECs 24 hours after administration of each siRNA. Two siRNAs (numbered 15 and 17) designed against C9orf169, a human R2R2 homologue, at concentrations of 5 and 20 nM, respectively, were selected based on their ability to downregulate C9orf169 expression in PBECs. C9orf169 expression was assessed by microarray analysis and RT-qPCR. The probe for C9orf169 is present on the Affymetrix HT HG-U219 Human Gene Expression Array. Graph (b) shows the downregulation of C9orf169 expression (assessed by microarray analysis) in PBECs 24 hours after administration of each siRNA.Administration of siRNA directed against the FAM25 family or VEGFA had no effect on C9orf169 expression. siRNA directed against R2R1 (human FAM25 family) and R2R2 (human C9orf169) downregulated KRT14 expression, a response similar to that observed after administration of siRNA directed against VEGFA and VEGF165. Knockdown of the FAM25 family and C9orf169 by siRNA resulted in downregulation of KRT14 gene expression (a basal cell marker). This response was most pronounced after administration of siRNA directed against C9orf169. Graph (c) shows the downregulation of KRT14 expression (normalized to PGK1) in PBECs 24 hours after administration of each siRNA. For comparison, siRNA directed against VEGFA and KRT14 are also included. [Figure 17] Schematic diagram of the effects of R2R homologs. Expression of R2R homologs leads to simultaneous regulation of HIF1A signaling (conferring oxygen tolerance) (Box 5) and a specific (PERP) anti-apoptotic pathway (Box 4). This allows for the (re)generation of robust epithelial cells (which possess a major defense barrier against stress) without conferring unlimited proliferative capacity. In other words, regulation of the specific anti-apoptotic pathway does not 'permit' general apoptosis resistance, which would lead to the dangerous situation of cell immortality, which would develop into cancer cells. This pathway was constructed based on microarray analysis of siRNA-mediated knockdown of human R2R1 homologs (FAM25 family) and human R2R2 homologs (C9orf169). [Figure 18A]R2R homologs are essential for 'keratinocyte regeneration and differentiation' (Box 1) and 'apoptosis regulation' (Box 2) in the VEGFA-VEGF165 pathway. The effects of R2R homologs on gene expression in 'keratinocyte regeneration and differentiation' (Box 1) are evident in Figures 18A and 18B. The effects of R2R homologs on gene expression in 'apoptosis regulation' (Box 2) are evident in Figure 18C. These are the net effects of Boxes 3, 4, and 5. [Figure 18B] R2R homologs are essential for 'keratinocyte regeneration and differentiation' (Box 1) and 'apoptosis regulation' (Box 2) in the VEGFA-VEGF165 pathway. The effects of R2R homologs on gene expression in 'keratinocyte regeneration and differentiation' (Box 1) are evident in Figures 18A and 18B. The effects of R2R homologs on gene expression in 'apoptosis regulation' (Box 2) are evident in Figure 18C. These are the net effects of Boxes 3, 4, and 5. [Figure 18C] R2R homologs are essential for 'keratinocyte regeneration and differentiation' (Box 1) and 'apoptosis regulation' (Box 2) in the VEGFA-VEGF165 pathway. The effects of R2R homologs on gene expression in 'keratinocyte regeneration and differentiation' (Box 1) are evident in Figures 18A and 18B. The effects of R2R homologs on gene expression in 'apoptosis regulation' (Box 2) are evident in Figure 18C. These are the net effects of Boxes 3, 4, and 5. [Figure 19A]A-B: The effect on cellular respiration (see Box 3 in Figure 17) is highly specific. siRNA-mediated knockdown of R2R homologs will downregulate oxidative phosphorylation. Expression of R2R homologs will upregulate oxidative phosphorylation. We can observe this effect most strongly in the expression of mitochondrial ATP5A1-ATP synthase (H+ transport) (Figure 19A). siRNA directed against R2R homologs will downregulate ATP5A1 expression. Figure 19B shows ATP5A1 expression within the context of global changes in gene expression of the oxidative phosphorylation pathway. [Figure 19B] A-B: The effect on cellular respiration (see Box 3 in Figure 17) is highly specific. siRNA-mediated knockdown of R2R homologs will downregulate oxidative phosphorylation. Expression of R2R homologs will upregulate oxidative phosphorylation. We can observe this effect most strongly in the expression of mitochondrial ATP5A1-ATP synthase (H+ transport) (Figure 19A). siRNA directed against R2R homologs will downregulate ATP5A1 expression. Figure 19B shows ATP5A1 expression within the context of global changes in gene expression of the oxidative phosphorylation pathway. [Figure 20]This figure shows the effects of R2R homologs on p53-p63 signaling (see Box 4 in Figure 17). PERP (a TP53 apoptotic effector) is downregulated by siRNA-mediated knockdown of the human R2R homolog. PERP is a p63-regulated gene essential for epithelial integrity. p63 is a master regulator of stratified epithelial development and is both necessary and sufficient to drive this multifaceted program. Perp, a four-transmembrane protein initially identified as an apoptosis-related target of the p53 tumor suppressor, is the first direct target of p63 specifically involved in this developmental program in vivo. This was demonstrated by Rebecca A. Ihrie et al. in 2005 (Cell, Vol. 120, 843-856, March 25, 2005) (italics refer to the authors in the abstract of this paper). The graph demonstrates downregulation of PERP expression after siRNA-mediated knockdown of the human R2R homologue. [Figure 21] FIG. 1 shows an overview of PERP expression within the overall changes in gene expression of the P53 pathway after administration of FAM25 nr20 (directed against the human homologue R2R1). [Figure 22] FIG. 1 shows an overview of PERP expression within the overall changes in gene expression of the P53 pathway after administration of FAM25 nr18 (directed against the human homologue R2R1). [Figure 23] FIG. 1 shows an overview of PERP expression within the global changes in gene expression of the P53 pathway after administration of C9orf169 nr15 (directed to the human homologue R2R2). [Figure 24] FIG. 1 shows an overview of PERP expression within the global changes in gene expression of the P53 pathway after administration of C9orf169 nr17 (directed against the human homologue R2R2). [Figure 25A]A-C: Expression of HIF1A (see Box 5 in Figure 17) is tightly regulated by the expression of human R2R homologs. siRNA-mediated knockdown of human R2R homologs down-regulates HIF1A expression (Figure 25A). The downstream effect of HIF1A on cellular respiration (oxidative phosphorylation) has been described (see Figure 17, Cellular Respiration). R2R homologs drive 'PERP-type p53-p63' and HIF1A expression. Thus, 'hardy' epithelial cells armed with HIF1A could acquire unlimited proliferative capacity under the influence of VEGFA / VEGF165. However, we simultaneously observed that epithelial cells are prevented from entering an immortal state. Expression of R2R homologs may act as a brake on the expression of genes that could render cells immortal. This is particularly evident in the case of the anti-apoptotic gene BCL2A1 (Figure 25B), whose expression confers resistance to therapy in cancer cells. Knockdown of R2R homologs by siRNA results in upregulation of BCL2A1 expression. R2R homologs act as a brake on BCL2A1 expression. R2R homologs also promote the expression of genes that allow cells to enter apoptosis when necessary. This is demonstrated by knockdown of R2R homologs by siRNA. This knockdown leads to downregulation of the tumor suppressor MAP2K4 (Figure 25C) in lung adenocarcinoma. [Figure 25B]A-C: Expression of HIF1A (see Box 5 in Figure 17) is tightly regulated by the expression of human R2R homologs. siRNA-mediated knockdown of human R2R homologs down-regulates HIF1A expression (Figure 25A). The downstream effect of HIF1A on cellular respiration (oxidative phosphorylation) has been described (see Figure 17, Cellular Respiration). R2R homologs drive 'PERP-type p53-p63' and HIF1A expression. Thus, 'hardy' epithelial cells armed with HIF1A could acquire unlimited proliferative capacity under the influence of VEGFA / VEGF165. However, we simultaneously observed that epithelial cells are prevented from entering an immortal state. Expression of R2R homologs may act as a brake on the expression of genes that could render cells immortal. This is particularly evident in the case of the anti-apoptotic gene BCL2A1 (Figure 25B), whose expression confers resistance to therapy in cancer cells. Knockdown of R2R homologs by siRNA results in upregulation of BCL2A1 expression. R2R homologs act as a brake on BCL2A1 expression. R2R homologs also promote the expression of genes that allow cells to enter apoptosis when necessary. This is demonstrated by knockdown of R2R homologs by siRNA. This knockdown leads to downregulation of the tumor suppressor MAP2K4 (Figure 25C) in lung adenocarcinoma. [Figure 25C]A-C: Expression of HIF1A (see Box 5 in Figure 17) is tightly regulated by the expression of human R2R homologs. siRNA-mediated knockdown of human R2R homologs down-regulates HIF1A expression (Figure 25A). The downstream effect of HIF1A on cellular respiration (oxidative phosphorylation) has been described (see Figure 17, Cellular Respiration). R2R homologs drive 'PERP-type p53-p63' and HIF1A expression. Thus, 'hardy' epithelial cells armed with HIF1A could acquire unlimited proliferative capacity under the influence of VEGFA / VEGF165. However, we simultaneously observed that epithelial cells are prevented from entering an immortal state. Expression of R2R homologs may act as a brake on the expression of genes that could render cells immortal. This is particularly evident in the case of the anti-apoptotic gene BCL2A1 (Figure 25B), whose expression confers resistance to therapy in cancer cells. Knockdown of R2R homologs by siRNA results in upregulation of BCL2A1 expression. R2R homologs act as a brake on BCL2A1 expression. R2R homologs also promote the expression of genes that allow cells to enter apoptosis when necessary. This is demonstrated by knockdown of R2R homologs by siRNA. This knockdown leads to downregulation of the tumor suppressor MAP2K4 (Figure 25C) in lung adenocarcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present invention is based on the discovery that two genes play important roles in tissue development and cancer biology. In particular, the inventors have found that these two genes are expressed in lung cells and are required for the late branching morphogenesis of lung epithelium and endothelium, sustaining the development / maintenance of the fine three-dimensional structure of the lung. These genes are essential for the squamous differentiation program and the development / maintenance of the progenitor cell pool (Krt14 expressing). Furthermore, the inventors have identified a crucial role for these genes in cancer biology, particularly in processes related to the acquisition of immortalization and metastatic phenotypes (including cancer progression and metastasis), as well as in cardiac development. We have identified the mouse and human sequences of these genes. Considering their coordinate expression and function as respiratory cell regeneration genes, we have identified these genes as R2R 1 and R2R 2 For simplicity, the term "R2R" will be used throughout most of this document. 1 / 2 ", which is a longer phrase "R2R 1 and / or R2R 2 " is used to represent

[0008] Therefore, R2R 1 / 2 When referring to genes, these names should be understood to include all mammalian forms of these genes, including human and rodent (mouse, rabbit, guinea pig, rat, etc.) forms. Furthermore, these names are intended to encompass all R2R 1 and / or R2R 2 In addition to including the sequences of the genes, it should be understood to also include fragments, portions, variants, derivatives and / or homologs / or orthologs of any of the genes described herein. In this regard, the term "R2R" 1 " should be understood to include the murine sequence encoded by the cDNA sequence designated 2200001I15Rik or RIKEN cDNA 2200001I15 and the seven human homologs designated FAM25A, FAM25B, FAM25C, FAM25D, FAM25E, FAM25G and FAM25HP. Furthermore, where appropriate, the term “R2R 1 / 2 " is R2R 1 and / or R2R 2 Includes the protein product of a gene or a fragment or portion thereof. In particular, the term “R2R 1 / 2 ", i.e., the term "R2R 1 " or "R2R 2 " includes any of the sequences set forth in SEQ ID NOs: 1 to 12 below, or fragments, portions, analogs, variants, or derivatives thereof.

[0009] Exemplary Mouse R2Rs 1 The sequence of the gene transcript is shown below in SEQ ID NO:1. SEQ ID NO: 1 acactgacacggaccgaaggagtggaaaaagctttacctgtcactgtctgctgccatacg ATGCTGGGAGGCCTGGGGAAGCTGGCGGCCGAGGGCCTGGCCCACCGCACAGAGAAAGCCACTGGGGGAGCAGTTCACGCAGTGGAAGAGGTGGTGAGCGAGGTGGTGGGCCACGCCAAGGAGGTTGGAGAGA AGACCATTAATGACGCCCTAAAGAAAGCCCAAGAATCAGGAGACAGGGTGGTGAAGGAGGTCACTGAGAAGGTCACCCACACCATCACTGATGCTGTTACCCATGCGGCAGAAGGCCTGGGAAGACTGGGACAG tgagcctgcctaccagcatggctggcccttcctgaaggtcaataaagagtgtgaaacgtgaaaaaaaaaaaaaaaataacaaaaaaaaaaaaaaaaaa

[0010] The portion of this sequence that encodes, or is translated from, this sequence is underlined and comprises approximately 267 nucleotides. This particular portion of SEQ ID NO:1 is set forth as SEQ ID NO:2. It will be apparent to one skilled in the art that the 267 nucleotides, when translated, give rise to a protein containing 89 amino acids, which has the following sequence (set forth in SEQ ID NO:3): SEQ ID NO: 3 MLGGLGKLAAEGLAHRTEKATGGAVHAVEEVVSEVVGHAKEVGEKTINDALKKAQESGDRVVKEVTEKVTHTITDAVTHAAEGLGRLGQ

[0011] Furthermore, the inventors have demonstrated that exemplary human R2R 1 The complete sequence of the gene transcript was determined and is shown below in SEQ ID NO:4. SEQ ID NO:4 actgtctgctgccacacg ATGCTGGGAGGCCTGGGGAAGCTGGCTGCCGAAGGCCTGGCCCACCGCACCGAGAAGGCCACCGAGGAGCCATTCATGCCGTGGAAGAAGTGGTGAAGGAGGTGGTGGGACACGCCAAGGAGACTGGAGAGA AAGCCATTGCTGAAGCCATAAAGAAAGCCCAAGAGTCAGGGGACAAAAAGATGAAGGAAATCACTGAGACAGTGACCAACACAGTCACAAATGCCATCACCCATGCAGCAGAGAGTCTGGACAAACTTGGACAG tgagtgcacctgctaccacggcccttccccagtctcaataaaaagccatgacatgtg The portion of this sequence that encodes, or is translated from, this sequence is underlined and comprises approximately 267 nucleotides. This particular portion of SEQ ID NO:4 is set forth in SEQ ID NO:5. It will be apparent to one skilled in the art that the 267 nucleotides, when translated, give rise to a protein containing 89 amino acids, which has the following sequence (set forth in SEQ ID NO:6): SEQ ID NO:6 MLGGLGKLAAEGLAHRTEKATEGAIHAVEEVVKEVVGHAKETGEKAIAEAIKKAQESGDKKMKEITETVTNTVTNAITHAAESLDKLGQ

[0012] Exemplary Mouse R2Rs 2 The sequence of the gene transcript is shown below in SEQ ID NO:7. SEQ ID NO:7 Gtgactggctgctgtctctagttgttgaggcctcttgggatctyggcgctmacmccwtgctytagwgactccgatagctcccrmggctccagtgsasmcctcggk cggnggnagggaaaaggcacttgctggtagctctgctcacccgcactgggacctggagctggaggactaagaagacagacggctgctgcttgccacagcctggacc ATGGACCCCCATGAGATGGTTGTGAAGAATCCATATGCCCACATCAGCATTCCTCGGGCTCACCTGCGCTCTGACCTGGGGCAGCAGTTAGAGGAGGTTCCTTCTTCATCTTCCTCCTCTGAGACTCAGCCTCTGCCTGCAGGAACATGTATCCCAGAGCCAGTGGGCCTCTTACAAACTACTGAAGCCCCTGGGCCCAAAGGTATCAAGGGCATCAAGGGTACTGCTCCTGAGCACGGCCAGCAGACCTGGCAGTCACCCTGCAATCCCTATAGCAGTGGGCAACGTCCATCGGGACTGACTTATGCTGGCCTGCCACCTGTAGGGCGTGGTGATGACATTGCCCACCACTGCTGCTGCTGCCCTTGCTGCTCCTGCTGCCACTGTCCTCGCTTCTGCCGTTGTCACAGCTGTTGTGTTATCTCC tagctgactattgaacctccagggctgtgcagcccaggttcctgctcaatgccaaagtgttgctggacatcaggagcagccgttgtcatgagcatcagccatttcctgcc ctgagcaggggagcctgtccaccagcgttcagctgtagccttctggaatagggttccagccactagccatgttggcaacaacagggacacccttcacgtcctgcaagact ttggcaataaagcaggatgagcgttgctgnncctgntgaaaanaaamwaaawacwgccgttgtcacarcygttrtgttatctmmkagstgacwattgtaammtycagrgc tgtrmagcccrggkksckgctcaatgccaaagtgttgmtgsmcmtcrggrgsrgccaagctttacgcggtacccgggatttttttgtacaaaaaggggccccctattagg

[0013] The portion of this sequence that encodes, or is translated from, this sequence is underlined and comprises approximately 426 nucleotides. This particular portion of SEQ ID NO:7 is set forth in SEQ ID NO:8. It will be apparent to one skilled in the art that the 426 nucleotides, when translated, give rise to a protein containing 142 amino acids, which has the following sequence (set forth in SEQ ID NO:9): SEQ ID NO:9 MDPHEMVVKNPYAHISIPRAHLRSDLGQQLEEVPSSSSSSETQPLPAGTCIPEPVGLLQTTEAPGPKGIKGIKGTAPEHGQQTWQSPCNPYSSGQRPSGLTYAGLPPVGRGDDIAHHCCCCPCCSCCHCPRFCRCHSCCVIS

[0014] Furthermore, the inventors have demonstrated that exemplary human R2R 2 The complete sequence of the gene transcript was determined and is shown below in SEQ ID NO:10. SEQ ID NO: 10 cttgaacccgggaggcagaggttgcagtgagccgagatcgcgcagctgcactccagcctgggcaacagagcaagactccatctcagaaaagaagcagaaagcctccaagagccaatggctc tcaagcatcttggtctctgctaagaagaggctcagaggcttagaagccctgcctcgccggggctttgaggtgtgtgagcaatggctggggactgcaggcccgggaatctgagggcctcaccc cacttcctttccagagccgtgacctcaggctcacctcctgccctcctctcaggcaagctgcagatgccctttagggcccaggccatgccccggatgtgaggggctgagtcactggtttggca gtgcccctcagagcccaggcctgggctgccacccacctgaggacgagggctgggccagctgtcgtgctccagttgctggggcctcttgggatcttgggaaccccatctctgagccccgcccc ATGGCCCCGCCCCTCCCAAGGAGGGAAAAGGCGGCTGCCAGTCGCTCAACTCAGGCACTGGGACCTAGAGCTCAGAAGACCGAGAGGACAGACTGCCGTGTTGCCACCACAGGCTGGACCATGGACCCCCAAGAGATGGTCGTCAAGAACCCATATGCCCACATCAGCATCCCCCGGGCTCACCTGCGGCCTGACCTGGGGCAGCAGTTAGAGGTGGCTTCCACCTGTTCCTCATCCTCGGAGATGCAGCCCCTGCCAGTGGGGCCCTGTGCCCCAGAGCCAACCCACCTCTTGCAGCCGACCGAGGTCCCAGGGCCCAAGGGCGCCAAGGGTAACCAGGGGGCTGCCCCCATCCAGAACCAGCAGGCCTGGCAGCAGCCTGGCAACCCCTACAGCAGCAGTCAGCGCCAGGCCGGACTGACCTACGCTGGCCCTCCGCCCGCGGGGCGCGGGGATGACATCGCCCACCACTGCTGCTGCTGCCCCTGCTGCCACTGCTGCCACTGCCCCCCCTTCTGCCGCTGCCACAGCTGCTGCTGCTGTGTCATCTCC tagcccagcccaccctgccagggccaggacccagacttcagcaaatgtggctcacacagtgccgggacatgccgggacatgcggggtggctgttgtcatgggcgtctgccccttcacaccaggcac tggggctcagacccaccaggaaggtggccgttcagcccgagctcctgaaacggaatcccaggtcctggctggagagggacacccctgattaccttaaggcccaggcaataaagcagggtgatcttc

[0015] The coding portion of this sequence, i.e., the portion from which this sequence is translated, is underlined and comprises approximately 552 nucleotides. This particular portion of SEQ ID NO:10 is set forth as SEQ ID NO:11. It will be apparent to one skilled in the art that the 552 nucleotides, when translated, give rise to a protein containing 184 amino acids, which has the following sequence (set forth in SEQ ID NO:12): SEQ ID NO: 12 MAPPLPRREKAAASRSTQALGPRAQKTERTDCRVATTGWTMDPQEMVVKNPYAHISIPRAHLRPDLGQQLEVASTCSSSSEMQPLPVGPCAPEPTHLLQPTEVPGPKGAKGNQGAAPIQNQQAWQQPGNPYSSSQRQAGLTYAGPPPAGRGDDIAHHCCCCPCCHCCHCPPFCRCHSCCCCVIS

[0016] Thus, the present invention relates to genes encoded by the sequences set out in SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10 and 11, as well as any fragments, portions, mutants, variants, derivatives and / or homologs / or orthologues thereof. Generally, the fragments, portions, mutants, variants, derivatives and / or homologs / or orthologues are functional or active, i.e., they are similar to wild-type R2R. 1 / 2 Maintains gene function.

[0017] The term "variant" can include naturally occurring variants as well as variants that are artificially created by introducing one or more nucleic acid additions, deletions, substitutions or inversions. Those skilled in the art will recognize that the human and mouse R2R sequences detailed above 1 / 2 It will be readily understood that genes homologous to the gene can be found in many different species, including, for example, other mammalian species. In some cases, homologous genes may only exhibit approximately 20 or 30% sequence homology or identity. In other cases, however, homologous genes may exhibit at least 40, 50, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology to the various nucleotide sequences set forth above. Thus, homologous genes from other species are intended to be within the scope of the present invention. Using the various nucleic acid and amino acid sequences described herein, one of skill in the art will readily be able to identify related sequences in other species, such as other mammals. For example, nucleic acids obtained from a particular species can be probed with the probes described herein for homologous or closely related sequences.

[0018] Furthermore, it should be understood that the present invention also relates to the products of the genes encompassed by the present invention, in particular the peptides encoded by SEQ ID NOs: 3, 6, 9 and 12. Furthermore, fragments, portions, analogs, variants, derivatives or homologous and / or identical proteins of any of these are also within the scope of the present invention. Generally, the fragments, portions, derivatives, variants and / or homologs are functional or active, i.e., they are functional or active relative to the wild-type R2R. 1 / 2 Maintains protein function. Additionally, proteins, polypeptides / peptides that are homologous / identical to any of the proteins encoded by SEQ ID NOs: 3, 6, 9, and 12 are also within the scope of the present invention. Protein or polypeptide / peptide sequences considered to be homologous or identical to any of the sequences described herein may exhibit only 20% or 30% sequence identity / homology. However, homologous / identical sequences may be at least 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% homologous or identical. Insofar as the present invention relates to fragments of any of the protein or polypeptide / peptide sequences described herein, it should be understood that fragments can comprise anywhere from about 10 amino acids to n-1 amino acids (where "n" is the number of amino acids in the entire sequence). For example, fragments can contain about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or about 125 amino acids (the maximum number of amino acids is determined by the number of amino acids in the entire sequence), and such fragments / portions can be referred to as peptide fragments. In one embodiment, the peptide fragments can be antigenic and / or immunogenic, i.e., they retain the ability to bind to antibodies that exhibit specificity, affinity, and / or selectivity for the native (intact) antigen, e.g., the antigen encoded by SEQ ID NOs: 3, 6, 9, and 12.

[0019] Those skilled in the art will recognize that R2R isolated from any given species may be useful for the various nucleic acid sequences and polypeptides described herein.1 / 2 It will be readily understood that natural variations, such as those due to polymorphisms, may exist between genes and proteins. Furthermore, it is known in the art that the degeneracy of the genetic code allows for the substitution of one or more bases in a codon without altering the primary amino acid sequence. Thus, genetic degeneracy can be exploited to obtain variant nucleic acid sequences that encode peptide or protein sequences that are substantially identical to the antigen sequences described herein. Indeed, the variant sequences provided by the present invention can be expressed as proteins and / or genes that exhibit one or more amino acid / nucleic acid substitutions, additions, deletions, and / or inversions relative to a reference sequence (e.g., any of the aforementioned sequences). Thus, it is to be understood that all such variants, particularly those which are functional or exhibit the desired activity, are within the scope of the present invention.

[0020] In other embodiments, the present invention provides a method for treating a R2R 1 / 2 The term "derivative" refers to any derivative of the R2R sequences described herein. 1 / 2 R2R, which contains one or more amino acid substitutions, deletions, additions, and / or inversions relative to the gene or peptide sequence 1 / 2 It may comprise a gene or peptide sequence. Additionally or alternatively, analogs of the various peptides described herein can be produced by introducing one or more conservative amino acid substitutions into their primary sequence. Those skilled in the art will understand that the term "conservative substitution" refers to the substitution of one or more amino acids of a protein or peptide with alternative amino acids having similar properties, without substantially altering the physicochemical properties and / or structure or function of the native (or wild-type) protein. Analogs of this type are also within the scope of the present invention. As is known in the art, the degeneracy of the genetic code allows for the substitution of one or more bases in a codon without changing the primary amino acid sequence. As a result, the sequences described herein are R2R 1 / 2Although known to encode proteins, the degeneracy of the code allows for variant nucleic acid sequences to be obtained that encode the same primary amino acid sequence.

[0021] As mentioned above, the present inventors have 1 / 2 It has been found that genes (and their protein products) are involved in pulmonary and cardiac morphogenetic events (e.g., cell signaling / migration, etc.) and sustain the development of the three-dimensional structures of the lungs and heart. Thus, a first aspect of the present invention provides R2R genes for use in medicine or for use in the treatment of diseases affecting cell / tissue development / structure, differentiation, proliferation and / or morphogenesis. 1 / 2 Genes and / or R2R 1 / 2 Providing proteins, e.g., R2R 1 / 2 Genes and / or R2R 1 / 2 The proteins can be used, for example, to treat lung and / or cardiac diseases and / or conditions, as well as cancer, particularly cancers affecting tissues of the lung or cardiac systems. In certain embodiments, the present invention provides R2R receptors for use in regulating cell transition events, such as events involved in mesenchymal-epithelial transition (MET) events and the reverse process, epithelial-mesenchymal transition (EMT). 1 / 2 Genes and / or R2R 1 / 2 Protein can be provided.

[0022] In a second aspect, the present invention provides a R2R agonist for their use in the treatment of pulmonary diseases and / or conditions or for their use in the manufacture of a medicament for the treatment of pulmonary diseases and / or conditions. 1 / 2 Genes and / or R2R 1 / 2 Provides protein. In another aspect, the present invention provides an R2R 1 In one embodiment, R2R genes and / or proteins are provided. 1The genes and / or proteins can be used in the treatment of diseases affecting the development / structure, differentiation, proliferation and / or morphogenesis of cardiac cells / tissues. In one embodiment, the present invention provides R2R ATPase inhibitors for use in the treatment of diseases and / or conditions affecting the development and / or formation of the ventricular septum. 1 Genes and / or proteins can be provided. In another aspect, the present invention provides an R2R antibody for use in the treatment of cancer. 1 In one embodiment, R2R genes and / or proteins are provided. 1 The genes and / or proteins can be used to treat cancers affecting various tissues, including, for example, lung and / or cardiac tissue. More generally, the present invention can be extended to the treatment of any cancer involving an abnormal / defective MET / EMT process. At least some examples of cancers that can be treated using the genes and / or proteins of the present invention are detailed below.

[0023] Again, the term “R2R 1 ", "R2R 2 " and "R2R 1 / 2 " includes not only the complete gene / peptide sequences referred to above, but also fragments, analogs, homologs, orthologs, variants and derivatives thereof.

[0024] The term "pulmonary disease" or "pulmonary condition" can include pulmonary pathologies, e.g., pathologies affecting lung development or the 3D structure of lung tissue. For example, "pulmonary disease" can include diseases and / or conditions affecting the epithelial cells lining the lung's airways, the endothelial cells of the lung's vascular network, and / or the differentiation and / or proliferation of these cells. Thus, pulmonary disease can include diseases affecting lung morphogenetic pathways and events. Pulmonary diseases and / or conditions affecting the differentiation of specific lung cell types (e.g., squamous epithelial cells) or the generation and / or maintenance of progenitor (basal) cell populations can also be treated by the compounds, medicaments, and methods described herein. Specific examples include diseases such as bronchopulmonary dysplasia (BPD) and / or chronic obstructive pulmonary disease (COPD), which can be treated using the compounds described herein. In other embodiments, the term "pulmonary disease" or "pulmonary condition" can include cell proliferation or neoplastic diseases, e.g., cancer, including non-small cell lung cancer (NSCLC) and / or small cell lung cancer (SCLC). The term "cardiac disease" or "cardiac condition" can include cardiac pathologies, such as pathologies affecting cardiac development or the 3D structure of cardiac tissue. Cardiac disease can include diseases that affect cardiac morphogenetic pathways and events, particularly mesenchymal-epithelial and epithelial-mesenchymal transitions. Specific examples include diseases such as atrial and ventricular septal defects, atrioventricular canal defects, malformations of the heart (atrioventricular) valves, and coronary arteries can be treated using the compounds described herein.

[0025] R2R as described herein 1 / 2 It should also be understood that because genes / proteins have been shown to be involved in morphogenetic events (e.g., cell signaling, etc.) and sustain the formation of the three-dimensional structure of complex tissues, such as the lung and / or heart, they can be applied to regenerative medicine. By way of example, when stem cells (e.g., adult embryonic or reprogrammed somatic cells (e.g., iPS cells) are used to repair or reconstruct damaged or diseased tissue, etc., the proteins and / or genes provided by the present invention can be used to facilitate tissue development. R2R1 / 2 These proteins and genes play an important role in pulmonary and cardiac morphogenesis as gatekeepers of epithelial-mesenchymal and mesenchymal-epithelial transitions. Therefore, they can be applied to cancer biology and cancer therapy. For example, diseases such as the transformation of localized cancer into cancer metastasis can be treated using the compounds described herein. R2R as described herein 1 / 2 In addition to providing various uses and medicaments comprising the genes and / or proteins, the present invention also provides methods of treating a subject suffering from any of the diseases and / or conditions described herein, including any of the cardiac / pulmonary diseases and / or conditions outlined above. Thus, a third aspect of the present invention is a method of treating a cardiac / pulmonary disease and / or condition, comprising administering to a subject in need thereof an R2R gene or gene product as described herein. 1 and / or R2R 2 Genes and / or R2R 1 and / or R2R 2 The method comprises administering a therapeutically effective amount of the protein.

[0026] Heart or lung disease or condition is R2R 1 / 2 A functional R2R as described herein when it results from or is associated with a lack or defect in gene and / or protein expression / function. 1 / 2 The use of the gene or protein may involve the interaction of normal (or wild-type) R2R to treat or alleviate the symptoms of a disease or condition. 1 / 2 It can provide a means to restore gene / protein function. The uses, medicaments and treatment methods described herein include recombinant R2R 1 / 2 It will be apparent that in some cases production of the gene / protein may be required. As such, the present invention further provides recombinant R2R 1 / 2 Consider methods for producing and / or expressing genes and / or proteins. PCR techniques can be used to generate R2R genes from various sources, such as lung tissue. 1 / 2It will be apparent to those skilled in the art that gene sequences can be selectively obtained. These sequences can be ligated to various expression control sequences or regulatory control sequences, such as promoters, operators, inducers, enhancer and / or silencer elements, ribosome binding sites, and / or terminator sequences. Those skilled in the art can select appropriate regulatory control sequences or expression control sequences for any given host. In another embodiment, PCR-derived R2R 1 / 2 The gene sequence can be introduced into a vector (e.g., a plasmid or expression cassette). In one embodiment, the vector can further comprise a tag or label nucleotide sequence to aid in protein purification methods.

[0027] The host cell is transformed with the vector and 1 / 2 Expression of gene sequences and recombinant R2R 1 / 2 The R2R can be maintained under conditions suitable to induce the production of R2R. 1 / 2 Vectors into which gene sequences (or fragments thereof) have been cloned can be introduced or transfected into cells using a variety of techniques. These techniques can also be called transfection protocols. Transfection protocols use conditions that make the cell membrane permeable to compounds such as nucleic acids. For example, electroporation, heat shock, chemical compounds (e.g., calcium phosphate, strontium phosphate), microinjection techniques, and / or gene guns can be used to facilitate the transfection of vectors, including expression vectors, into cells. Techniques used to purify recombinant proteins so produced are known, and where the recombinant protein is tagged or labeled, these techniques can include the use of affinity chromatography techniques and the like. In view of the above, the fourth and fifth aspects of the present invention provide R2R 1 / 2 Expression vectors containing the gene sequences and host cells transformed therewith are provided.

[0028] R2R as a means of treating various diseases and / or conditions (e.g., cardiac and / or pulmonary diseases and / or conditions) 1 / 2 In addition to providing genes and / or proteins, the present invention also provides R2R 1 / 2 It can regulate gene expression and R2R 1 / 2 Provided are compounds that can be useful for treating conditions resulting from or associated with the overexpression of genes / proteins. Such compounds can be oligonucleotides, preferably antisense oligonucleotides, which can be in the form of, for example, DNA and / or RNA. In one embodiment, the oligonucleotide is an RNA molecule known to those skilled in the art as a small / short interfering and / or silencing RNA, hereinafter referred to as siRNA. Such siRNA oligonucleotides can be in the form of natural double-stranded RNA or double-stranded RNAs that have been modified in some way (e.g., by chemical modification) to be nuclease-resistant. Additionally or alternatively, siRNA oligonucleotides can be in the form of short hairpin RNA (shRNA) expression or plasmid constructs that correspond to or contain the siRNAs described herein.

[0029] The oligonucleotides provided by the present invention are R2R 1 / 2 They can be designed to regulate the expression of genes. 1 / 2 By analyzing the sequences and utilizing algorithms such as BIOPREDsi, one skilled in the art can easily determine or predict by computer the nucleic acid sequences that will show optimal knockdown effects for these genes (see, for example, http: / / www.biopredsi.org / start.html). Thus ... 1 / 2 They can be produced and tested to determine whether they are capable of modulating the expression of the gene. In view of the above, the antisense oligonucleotides and / or siRNA molecules described herein can be used for (i) the treatment of any of the diseases and / or conditions described herein, in particular (ii) the treatment of pulmonary diseases and / or disorders, (iii) the treatment of cardiac diseases and / or conditions, and (iv) the treatment of cancer. Furthermore, the antisense oligonucleotides and / or siRNA molecules described herein can be used in the manufacture of a medicament for treating the diseases outlined in (i)-(iv) above, or in methods of treating a subject suffering from such diseases and / or disorders.

[0030] Furthermore, R2R 1 / 2 Antibodies (or antigen-binding fragments thereof) capable of binding to the protein may be useful in treating the diseases and / or conditions described herein, including, for example, cardiac and / or pulmonary diseases and / or conditions. 1 / 2 Antibodies that block or neutralize the function of proteins are useful in treating diseases and / or conditions that are R2R 1 / 2 It can be particularly useful when produced by overexpression of a protein. Techniques used to produce monoclonal antibodies (mAbs) are well known, and R2R 1 and / or R2R 2 These antibodies can be readily utilized to produce mAbs specific to either proteins or their fragments. Similarly, the processes used to produce polyclonal antibodies are also well established and R2R 1 and / or R2R 2 It can be used to generate antibodies specific to either the protein or a fragment thereof. Other compounds useful in treating the diseases and / or conditions described herein (e.g., cardiac and / or pulmonary conditions and / or disorders or cancer) can include, for example, proteins, peptides, amino acids, carbohydrates, and other small organic molecules. In addition to the above, isolated R2R 1 / 2The nucleotide and / or protein sequences can be used as a basis for the design of probes and / or primers for use in ex vivo and / or in situ detection and expression studies. Typical detection studies include, for example, polymerase chain reaction (PCR), hybridization studies, sequencing protocols, and immunological and / or Southern / Northern blotting detection techniques. In said detection and / or expression tests, in principle any polynucleotide (or oligonucleotide) or polypeptide fragment designed from the aforementioned sequences can be used.

[0031] Generally, polynucleotide fragments for use as probes and / or primers contain 10-30 nucleotides (although other lengths may be useful for particular applications), exhibit some degree of specificity for particular sequences, and do not bind to unrelated sequences. Similarly, polypeptide fragments used as probes can also be relatively short, generally containing 5-20 amino acids (although other lengths, shorter or longer, may be useful for some applications). It will be readily appreciated that by careful selection of primer / probe sequences and the use of stringent (preferably highly stringent) hybridization conditions, any non-selective binding will be minimized. Thus, oligonucleotide probe and / or primer sequences having at least 50%, at least 75%, at least 90% or at least 95% complementarity to all or a portion of the nucleotide sequences set forth herein, as well as those having exact (i.e., 100%) complementarity thereto, should be considered within the scope of the present invention.

[0032] Hybridization between probes / primers and nucleic acid sequences (such as any described herein) can be achieved in 2-6 x SSC (i.e., 2-6 x NaCl 17.5 g / L and sodium citrate (SC) 8.8 g / L) buffered saline containing 0.1% sodium dodecyl sulfate (SDS) at temperatures between about 40 °C and 75 °C. Of course, depending on the degree of similarity between the probe / primer and the sequence, buffers with decreasing SSC concentrations (i.e., 1 x SSC with 0.1% SDS, 0.5 x SSC with 0.1% SDS, and 0.1 x SSC with 0.1% SDS) can also be used. Polypeptide probes having at least 30%, 50%, 70%, 75%, 80%, 85%, 90% or 95% identity to all or a portion of the amino acid sequences disclosed herein, as well as those having exact (i.e., 100% identity) thereto, should be considered within the scope of the present invention.

[0033] Thus, a further aspect of the invention provides oligonucleotide probes and / or primers designed to hybridize to all or part of a sequence selected from the group consisting of SEQ ID NOs: 1; 2; 4; 5; 7; 8; 10 and 11. Additionally, a further aspect provides polypeptide probes designed to bind to all or part of a sequence selected from the group consisting of SEQ ID NOs: 3; 6; 9 and 12. In another aspect, the invention provides a method for diagnosing a pulmonary disease or condition and / or susceptibility thereto, comprising: 1 and / or R2R 2 determining whether the gene is aberrantly expressed. Subjects diagnosed with cancer and / or heart and / or lung diseases and / or conditions, etc., may have abnormal (i.e., increased or decreased) R2R 1 and / or R2R 2The expression of a gene / protein can be indicated. The term "aberrant expression" should be understood to include increased and / or decreased gene expression levels relative to expression in a sample from a healthy subject or a subject not suffering from a disease and / or condition (i.e., a cardiac or pulmonary disease and / or condition or cancer).

[0034] The term "sample" should be understood to include samples of bodily fluids, such as whole blood, plasma, serum, saliva, sweat, and / or semen. In other examples, "samples" such as tissue biopsies and / or scrapings can be used. In particular, lung tissue biopsies and / or scrapings can be used. Furthermore, samples can include tissue or glandular secretions, and lavage protocols can be used to obtain fluid samples secreted into the lungs, etc. Suitable lavage protocols can include bronchoalveolar lavage procedures. Such samples can be prepared by subjecting a large amount of R2R 1 / 2 Nucleic acids (i.e., DNA or RNA) and / or R2R 1 / 2 It will be apparent to one skilled in the art that the methods can provide proteins, peptides (or fragments thereof). Furthermore, these methods can include an initial step of providing a sample from a subject suspected of suffering from or at risk of developing a pulmonary disease and / or condition.

[0035] R2R 1 and / or R2R 2 Increased gene / protein expression levels can be associated with any of the aforementioned diseases and / or conditions or susceptibility thereto. For example, R2R 1 / 2 Increased gene / protein expression can indicate excessive cell proliferation and / or differentiation and can be associated with neoplastic conditions, such as cancer (i.e., lung cancer). 1 and / or R2R 2Decreased gene / protein expression can indicate a pathological condition characterized by poor or impaired cardiac / pulmonary development, which can result in tissue damage (due to mechanical stress acting within the cardiac / pulmonary system), scarring, loss of cardiac / pulmonary structural integrity, and deformation or damage to the pulmonary airways or cardiac structures. Proteins and / or genes in a sample, such as the above-mentioned sample, e.g., R2R 1 / 2 Techniques that can be used to identify gene and / or protein levels will be apparent to those skilled in the art.

[0036] Such techniques may include, for example, polymerase chain reaction (PCR) based techniques such as real-time PCR (also known as quantitative PCR). In this example, real-time PCR is used to determine R2R 1 and / or R2R 2 The expression level of a gene encoding a protein can be determined. Generally, and to quantify the expression level of a specific nucleic acid sequence, reverse transcriptase PCR can be used to reverse transcribe mRNA associated with a complementary DNA (cDNA). Preferably, the reverse transcriptase protocol can use primers designed to specifically amplify the mRNA sequence of interest. PCR can then be used to amplify the cDNA generated by reverse transcription. Typically, cDNA is amplified using primers designed to specifically hybridize to particular sequences, and the nucleotides used in PCR can be labeled with fluorescent or radiolabeled compounds. Techniques for using labeled nucleotides to allow quantitation of the amount of DNA produced during PCR will be apparent to those skilled in the art. Briefly, by way of example, the amount of labeled amplified nucleic acid can be determined by monitoring the amount of labeled nucleotide incorporated during PCR cycling. Further information regarding the PCR-based techniques described herein can be found, for example, in PCR Primer: A Laboratory Manual, Second Edition Edited by Carl W. Dieffenbach & Gabriela S. Dveksler: Cold Spring Harbour Laboratory Press and Molecular Cloning: A Laboratory Manual by Joseph Sambrook & David Russell: Cold Spring Harbour Laboratory Press.

[0037] R2R in the sample 1 and / or R2R 2 Other techniques that can be used to determine the expression level of a gene include, for example, Northern and / or Southern blot techniques. Northern blots can be used to determine the amount of a particular mRNA present in a sample, and as such, R2R 1 and / or R2R 2 The expression level of the gene can be determined. Briefly, mRNA can be isolated using techniques known to those skilled in the art, for example, by isolating an expressible R2R 1 and / or R2R 2 The gene can be extracted from a cell-based or cell-free system engineered to contain the gene and subjected to electrophoresis. A nucleic acid probe designed to hybridize to (i.e., complementary to) the mRNA sequence of interest, in this example, R2R 1 and / or R2R 2 The mRNA that encodes the protein can then be used to detect and quantitate the amount of that particular mRNA present in a sample. Additionally or alternatively, R2R 1 and / or R2R 2 The expression levels of genes can be identified by microarray analysis. Such methods are useful for R2R 1 and / or R2R 2This would involve the use of DNA microarrays containing nucleic acids derived from genes. 1 and / or R2R 2 To identify the expression level of a gene, one skilled in the art can extract nucleic acid, preferably mRNA, from a system (cell-based or cell-free) that has been subjected to the method described in the first aspect of the invention, and subject it to an amplification protocol, such as reverse transcriptase PCR, to generate cDNA. Preferably, a specific mRNA sequence, in this example R2R 1 and / or R2R 2 Primers specific to the gene coding sequence can be used.

[0038] Amplified R2R 1 and / or R2R 2 The cDNA can be subjected to a further amplification step, optionally in the presence of labeled nucleotides (as described above). The labeled and amplified cDNA can then be contacted with a microarray under conditions that allow binding to the DNA of the microarray. In this way, R2R 1 and / or R2R 2 The expression level of the gene can be identified. Further information on the above techniques can be found, for example, in PCR Primer: A Laboratory Manual, Second Edition Edited by Carl W. Dieffenbach & Gabriela S. Dveksler: Cold Spring Harbour Laboratory Press and Molecular Cloning: A Laboratory Manual by Joseph Sambrook & David Russell: Cold Spring Harbour Laboratory Press. R2R in the sample 1 / 2 To determine the levels of the protein, 1 / 2 Immunological techniques can be used which utilize agents capable of binding to proteins.

[0039] In one embodiment, the aforementioned diagnostic method comprises combining the support (or a portion thereof) with the test sample to determine any R2R present in the sample. 1 / 2 The step can include contacting under conditions that allow for the association, interaction, binding and / or immobilization of proteins to said support. Suitable supports may include, for example, glass, nitrocellulose, paper, agarose and / or plastic. Supports such as plastic materials may take the form of a microtiter plate. Alternatively, the support that is contacted with the test sample may be a R2R 1 / 2 The compound may comprise an agent capable of binding to the protein(s). Preferably, the compound is an R2R 1 / 2 The agent capable of binding to the protein is bound to the support (or at least a portion thereof). Suitable binding agents are, for example, R2R 1 / 2 It can include antibodies, such as monoclonal or polyclonal antibodies, and / or other types of peptides or small molecules that can bind to proteins. It should be understood that this definition applies to all types of binding agents described herein. Thus, the support (or a portion thereof) can be an R2R 1 / 2 A drug capable of binding to a protein and any R2R present in the sample 1 / 2 Contact with the test sample can be under conditions that allow binding or interaction between the proteins.

[0040] R2R 1 / 2 Any R2R bound to a support or agent capable of binding to a protein(s). 1 / 2 Proteins are R2R 1 / 2 Detection can be achieved using an additional agent (hereinafter referred to as a "primary binding agent") capable of binding to the protein(s). Additionally or alternatively, the primary binding agent can be an R2R 1 / R2R 2 Protein:support complex or R2R 1 / 2 Proteins and R2R 1 / 2It has affinity for or is capable of binding to the above agents capable of binding to proteins. The primary binding agent can be conjugated to a moiety that allows it to be detected (hereinafter referred to as a "detectable moiety"). For example, the primary agent can be conjugated to an enzyme that reports levels via a colorimetric chemiluminescent reaction. Such conjugated enzymes include, but are not limited to, horseradish peroxidase (HRP) and alkaline phosphatase (AlkP). Additionally or alternatively, the primary binding agent can be conjugated to a fluorescent molecule, such as a fluorophore, such as FITC, rhodamine, or Texas Red. Other types of molecules that can be conjugated to the binding agent include radiolabeled moieties.

[0041] Or, R2R 1 / 2 Any R2R bound to a support or agent capable of binding to a protein(s). 1 / 2 The protein can be detected using a further binding agent (hereinafter referred to as a "secondary binding agent") that has affinity for the primary binding agent. Preferably, the secondary binding agent is conjugated to a detectable moiety. R2R 1 / 2 The amount of primary binding agent bound to the protein(s) (or secondary binding agent bound thereto) is determined by the R2R 1 / 2 The level of the protein(s) can be expressed. In one embodiment, R2R 1 / 2 The method for identifying the level of a protein comprises: combining a support (or a portion thereof) with a test sample to identify any R2Rs present in the sample; 1 / 2 This can take the form of a "dipstick" test in which the protein(s) are contacted under conditions that allow binding to a support or binding agent to which they are bound or immobilized. In other embodiments, the method can take the form of an immunological assay, such as an enzyme-linked immunosorbent assay (ELISA). An ELISA involves contacting a test sample with a support and detecting any R2R present in the sample. 1 / 2The protein(s) are attached to a binder (R2R 1 / 2 Alternatively, the sample can be "captured" or bound by any R2R present in the sample. 1 / 2 The protein(s) can be contacted with the support under conditions that allow for "direct" binding between the protein(s) and the support.

[0042] Each of the aforementioned ELISA methods is a "direct" R2R 1 / 2 A protein detection step or "indirect" identification step may be included. ELISAs that include such steps are sometimes known as "direct" or "indirect" ELISAs. "Direct" ELISA involves connecting the test sample and the support to any R2R present in the sample. 1 / 2 This can include contacting the protein(s) under conditions that allow binding of the protein(s) to the support and / or binding agent therefor. After an optional blocking step, the bound R2R 1 / 2 The protein(s) are R2R 1 / 2 Detection can be achieved using an agent capable of binding to the protein (i.e., a primary binding agent), preferably conjugated to a detectable moiety. "Indirect" ELISA is R2R 1 / 2 After contacting the protein(s) with the primary binding agent, a further step can be included using a further binding agent (secondary binding agent) that has affinity or specificity for the primary binding agent. Preferably, the secondary binding agent can be conjugated to a detectable moiety.

[0043] R2R in the sample 1 / 2 Other immunological techniques that can be used to identify protein levels include the use of binding agents, e.g., R2R 1 / 2 An antibody capable of binding to the protein(s) is / are attached to any R2R present in the sample. 1 / 2 Protein(s) and R2R 1 / 2Immunohistochemistry involves contacting a sample, preferably a tissue sample, with a protein-binding agent under conditions that allow binding between the agent and the sample. Typically, the sample is treated with a detergent, such as Triton X100, before contacting the sample with the binding agent. Such techniques can be referred to as "direct" immunohistochemistry. Alternatively, the test sample may be a roll-to-roll (R2R) sample. 1 / 2 After contact with a protein-binding agent, R2R 1 / 2 R2R is performed using an additional binding agent (secondary binding agent) that is specific for, has affinity for, or is capable of binding to the protein binding agent. 1 / 2 The cells can be subjected to an indirect immunohistochemical staining protocol to detect protein / binding agent complexes. It will be apparent to those skilled in the art that in both direct and indirect immunohistochemistry techniques, the binding agent or secondary binding agent can be conjugated to a detectable moiety. Preferably, the binding agent or secondary binding agent is conjugated to a moiety that can report the level of bound binding agent or secondary binding agent by a colorimetric chemiluminescent reaction.

[0044] R2R present in the sample 1 / 2 The results of the immunohistochemical staining can be compared to the results of immunohistochemical staining performed on a control sample to identify the level of the protein(s). By way of example, a sample showing more or less bound protein binding agent (or secondary binding agent) than the control sample can be one provided by a subject with a particular disease and / or condition. R2R 1 / 2 Other techniques that utilize the use of agents capable of binding to proteins include, for example, techniques such as Western blots or dot blots. Western blots can involve subjecting a sample to electrophoresis to separate or resolve components, such as protein components of the sample. The resolved components can then be transferred to a support, such as nitrocellulose. Any R2R present in the sample can be resolved. 1 / 2 To identify the protein(s), any R2R present in the sample 1 / 2Protein(s) and R2R 1 / 2 R2R the support under conditions that allow binding between the agent capable of binding to the protein(s). 1 / 2 It can be contacted with a binding agent capable of binding to the protein(s).

[0045] Conveniently, R2R 1 / 2 The agent capable of binding to the protein(s) can be conjugated to a detectable moiety. Alternatively, the support may be a R2R 1 / 2 The binding agent(s) capable of binding to the protein(s) may be contacted with a further binding agent that has affinity for the binding agent(s). Conveniently, the further binding agent may be conjugated to a detectable moiety. In the case of a dot blot, the sample or a portion thereof is analyzed to determine whether any R2R 1 / 2 The protein(s) can be contacted with the support such that they are bound or immobilized to the support. 1 / 2 Identification of the protein(s) can be carried out as described above. In any of the above techniques, the amount of primary or secondary binding agent detected can be determined by the R2R 1 / 2 Furthermore, results obtained from all or part of the diagnostic methods described herein can be compared to results obtained from reference or control samples from healthy subjects known not to be suffering from or susceptible to a particular disease or disorder (such as a cardiac and / or pulmonary disease or disorder and / or cancer).

[0046] A further aspect of the present invention is R2R 1 and / or R2R 2 A method for identifying or producing an agent that modulates the expression of a gene, comprising: 1 and / or R2R 2 The gene is contacted with the test drug, and R2R 1 / 2The method further comprises detecting any modulation of gene expression. The methods, such as those described in this ninth aspect of the invention, may be used in a system, such as a R2R 1 and / or R2R 2 It will be apparent to one skilled in the art that this can be carried out in cell-based systems or cell-free systems, such as cells engineered to contain the R2R gene. 1 or R2R 2 The cells can be transfected with a nucleic acid comprising any of the genes. In one embodiment, the nucleic acid can be in the form of a vector (e.g., a plasmid or expression cassette as described above).

[0047] In one embodiment, the results obtained from the aforementioned method are R2R 1 and / or R2R 2 The results can be compared to those obtained from a control where the gene is not contacted with the test agent. In this way, it can be determined whether the agent is R2R 1 and / or R2R 2 It is possible to determine whether the expression of a gene can be regulated. 1 and / or R2R 2 If the expression level of the gene is lower than or higher than the expression level detected in the control method, the test agent is R2R. 1 and / or R2R 2 If the expression level is the same as that observed in the control method, the test agent may be useful as an R2R modulator of the gene. 1 and / or R2R 2 It is assumed that the expression of the gene cannot be regulated. Suitable test agents can take the form of nucleic acids, such as the antisense oligonucleotides mentioned above, proteins, peptides, amino acids, antibodies (and fragments thereof), carbohydrates and other small organic molecules. In a further aspect, the present invention provides a method for treating a rhodium-containing ... 1 / 2a gene / protein, an antisense oligonucleotide (DNA or RNA) as described herein, and / or an R2R gene identified by the method provided by the ninth aspect of the invention; 1 and / or R2R 2 Pharmaceutical compositions containing any of the agents capable of regulating gene / protein expression or function are provided. Such compositions can be applied to the treatment of various diseases and / or conditions described herein, including, for example, heart and / or lung diseases and / or the aforementioned cancers.

[0048] Preferably, the pharmaceutical composition provided by the present invention is formulated as a sterile pharmaceutical composition. Suitable excipients, carriers or diluents can include, for example, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, aqueous salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium silicate, polyvinylpyrrolidone, cellulose-based substances, polyethyleneglycone, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polypropylene-block polymers, polyethylene glycol and wool fat, etc., or combinations thereof. The pharmaceutical preparation can be formulated in a form suitable for oral, parenteral, or topical administration, for example. In one embodiment, the pharmaceutical composition can be formulated so that it can be inhaled. The composition administered by inhalation can be in the form of a fine powder or solution that can be aerosolized and inhaled as droplets. Those skilled in the art will be familiar with devices that can be used to deliver compositions directly to the lungs, such as by inhalation. The droplet or particle size of the composition can be varied to allow the drug to reach various regions of the lungs. For example, once inhaled, small particles or droplets can reach deep lung tissue and, in some cases, the alveoli. Pharmaceutical compositions formulated for topical administration may be presented as an ointment, solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

[0049] R2R 1 and / or R2R 2 A compound capable of regulating a gene, e.g., a compound identified by the methods described herein or an R2R described herein 1 / 2 Gene / protein fragments, antisense oligonucleotides, antibodies, and the like may find further use as modulators of cell differentiation. 1 and R2R 2 The genes and their products are expressed in lung epithelial cells, particularly squamous epithelial cells arising from lung epithelial progenitor cells or basal cell populations (i.e., Krt14 +ve We have confirmed that it is involved in pathways that regulate differentiation of mitochondrial cells. Compounds that modulate cell differentiation (e.g., lung epithelial cells) can be particularly useful in treating disorders such as BPD, which results in damaged, scarred lungs that exhibit poor or insufficient regenerative capacity. Thus, administering or using compounds that modulate cell differentiation can improve or restore the regenerative capacity of the lungs. 1 and / or R2R 2Those skilled in the art will readily appreciate that compounds that can enhance or promote the expression of genes or restore the function of these genes can be particularly useful.

[0050] In another aspect, the present invention provides animal models for studying tissue development and / or cell migration events, as well as certain diseases and / or conditions, including cardiac and / or pulmonary diseases and / or conditions and cancer. In one embodiment, the animal model is R2R 1 / 2 Additionally or alternatively, animal models can be created by manipulating or modulating (i.e., upregulating or downregulating) the expression of genes / proteins. 1 / 2 As mentioned above, many human and mouse R2R 1 / 2 Disclosure of the gene / protein sequences ensures that one skilled in the art can easily manipulate / adjust these sequences to create animal models in situ, e.g., "knockout" animals, i.e., R2R 1 / 2 Animals can be generated in which expression of gene sequences is reduced or substantially eliminated. Such models are useful for testing the effects of drugs potentially useful in treating the diseases and / or disorders described herein and / or for determining the function or role of particular genes. Alternatively, R2R 1 / 2 Animal models can also be created in which gene / protein expression is upregulated. In this way, upregulated R2R 1 / 2 Drugs aimed at suppressing gene / protein expression can be tested for efficacy and function. 1 / 2 It is also possible to study the effect of gene protein expression.

[0051] In other embodiments, R2R domains are used to alter the activity of the protein and aid in elucidating the function of particular domains, amino acids, etc. 1 / 2Substitutions, additions, deletions and / or inversions can be introduced into the gene sequence. Additionally or alternatively, knockout animals, such as those described above, can be transformed with any of the gene sequences described herein. This can result in variant or mutant R2R 1 / 2 It is particularly useful when the effect of a gene or the efficacy of a drug or compound that can inhibit the expression or function of said variant or mutated sequence is to be studied.

[0052] Table 1. RIKEN cDNA 2200001I15 gene and RIKEN cDNA 2310002J15 gene among the top list of genes in SAM analysis of differential gene expression at E16.5 in wild-type and Vegf120 / 120 knockout anti-cytokeratin 4-5-6-8-10-13-18 stained cells. TIFF0007723057000001.tif166166

[0053] Table 2. RIKEN cDNA 2200001I15 gene and RIKEN cDNA 2310002J15 gene among the top list of genes in SAM analysis of differential gene expression at E16.5 in wild-type and Vegf120 / 120 knockout GS-IB4 stained cells. TIFF0007723057000002.tif250138 TIFF0007723057000003.tif234147 TIFF0007723057000004.tif249130 TIFF0007723057000005.tif249142 TIFF0007723057000006.tif24689

[0054] Table 3. Distribution of embryos by gestational age and Vegf genotype status TIFF0007723057000007.tif195157 TIFF0007723057000008.tif69157wt / wt = homozygous wild type (Vegf+ / +), 120 / wt = heterozygous Vegf120 / +, 120 / 120 = homozygous Vegf120 / 120 knockout (NG = genotype not determined due to insufficient embryological morphology)

[0055] Overview In a VEGF mouse knockout model, we found R2R genes, transcripts, and their respective proteins. Vegf120 / 120 knockout mice lack Vegf 164 and Vegf 188 Inability to produce isoforms (Vegf 164 is human VEGF 165 The lungs of Vegf120 / 120 knockout mice are underdeveloped at birth, and peripheral airway and vascular differentiation are severely impaired in the lungs of Vegf120 / 120 knockout embryos. 164 and VEGF in humans 165 This led to the discovery that the basal cells drive a very specific gene expression program. This program has two components. The first component is the (re)generation of basal cells of the airway epithelium. Basal cells are the origin of at least the cell population of the proximal airway. Basal cells also generate strong intercellular junctions by laying down hemidesmosomes and focal adherens junctions. Basal cells reinforce their intracellular structure with the intermediate filament proteins KRT14 and KRT5. These two proteins are attached to (hemi)desmosomes.

[0056] The second component is the differentiation program. This is the 'strengthening program' ('squamous differentiation') of the cells lining the airways. These cells need to be robust at birth because they are exposed to mechanical stress and high levels of oxygen. This strengthening is made possible by a family of proteins that strengthen the cellular structure. This protein family consists of intermediate filaments and proteins that strengthen these intermediate filaments (e.g., SPRR proteins, LORs, HRNRs). These two programs can be summarized as follows: 'keratinocyte differentiation', 'epidermal cell differentiation', 'intermediate filament remodeling', 'cornified envelope', 'keratinocyte differentiation', and 'cytoskeleton remodeling keratin filaments'.

[0057] VEGF for regeneration of injured lung in humans 165 Although using proteins is attractive, VEGFA and VEGF 165 is a key regulator of a wide variety of processes in this organism. 165 administration would have too many side effects. Vegf in mice 164 and VEGF in humans 165 In a gene expression program driven by 1 and R2R 2 These novel genes, their transcripts, and translated proteins are unrelated to genes and their downstream products of the basal and squamous gene expression programs. We conducted experiments to determine whether these genes are important modulators of the basal and squamous differentiation programs. One of the most important findings from these experiments was that these genes are important modulators of HIF1α and PERP expression in cells. Our experiments showed that R2R genes are positive regulators, and disrupting this mechanism opens up therapeutic possibilities for cancer treatment.

[0058] material and method Mouse embryo and tissue preparation All animal experiments were approved by the Animal Ethics Committee of Leiden University Medical Center and conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Heterozygous Vegf+ / 120 mice were mated to generate Vegf120 / 120 embryos and Vegf+ / + wild-type littermates. Embryonic day (E) 0.5 was defined as the morning of the day a vaginal plug was observed. Pregnant females were sacrificed by cervical dislocation. E12.5, 14.5, and 16.5 embryos were isolated in sterile PBS. The thorax of the embryos was carefully dissected under RNase-free conditions, placed in tissue freezing medium (TBS, Triangle Biomedical Sciences, Durham, NC), frozen, and stored at -80°C. The distribution of embryos according to gestational age and maternal origin is shown in Table S1. Cryostat sections (8 μm) were cut and adhered to SuperFrost Plus microscope slides (Menzel GmbH & Co KG, Braunschweig, Germany). Sectioning and further immunohistochemical processing of thoraxes of embryos of different embryonic ages was performed randomly.

[0059] Immunohistochemistry and laser capture microdissection Three tissue sections were selected from the rib cage of each embryo at the biventricular level of the heart. These were processed for immunohistochemistry in a single batch. Cryostat sections were fixed by placing the slides in cold acetone (4°C) within 2 minutes of removal from the -80°C freezer. All further immunohistochemistry steps were performed at 4°C, and all buffers and antibody solutions were kept at 4°C. RNase-free PBS or D-PBS buffer was prepared by diluting RNAsecure (25x, AM7006, Ambion, TX) to 1x with the desired buffer. All antibody solutions were prepared in PBS, except for the isolectin GS-IB4 complex, which was diluted in D-PBS. Superase.In (AM2696, Ambion, Austin, TX) was added to each antibody solution at a final concentration of 1 U / μl. Slides were air-dried, and tissue sections were outlined with a hydrophobic barrier pen. After placing the slides on a cold metal block (4°C), 30 μl of PBS was applied to each tissue section and allowed to drain. Subsequently, 30 μl of mouse anti-pan keratin (4, 5, 6, 8, 10, 13, 18) monoclonal antibody (MAB1636, Chemicon) at a concentration of 10 μg / 100 μl was added dropwise to the sample. After 2 minutes, the antibody solution was removed, and the tissue sections were gently rinsed with 250 μl of PBS. Next, 30 μl of Alexa-fluor-488 chicken anti-mouse IgG (H+L) conjugate (A21200, Invitrogen, CA) at a concentration of 10 μg / 100 μl was applied for 2 minutes, followed by another gentle wash with 250 μl of PBS. Finally, the staining procedure was completed with a third cycle of 2 min of staining with 30 μl of isolectin GS-IB4 Alexa Fluor 594 conjugate (I21413, Invitrogen, CA) at a concentration of 10 μg / 100 μl. Tissue sections were dehydrated at room temperature in 75% EtOH (30 s), 95% EtOH (30 s), 100% EtOH (30 s), 100% EtOH (120 s), and xylene (180 s). Immediately after the dehydration step, laser capture microdissection was performed using a Veritas Microdissection Instrument (Arcturus Bioscience, Mountain View, CA).We dissected 3 x 300-400 cells (in triplicate samples) from intrapulmonary airways or blood vessels in the embryonic lungs of three tissue sections at the biventricular level of the heart. Cell staining with a mouse anti-pan keratin monoclonal antibody / chicken anti-mouse IgG Alexa-fluor-488 conjugate identified them as green fluorescent cells (blue filter). These green fluorescent cells represented airway epithelial cells (ker+ cells) and were randomly dissected, regardless of their proximal or distal airway morphology. Cell staining with the isolectin GS-IB4 Alexa-fluor-594 conjugate identified them as red fluorescent cells (green filter). These cells were defined as mesenchymal cells with endothelial characteristics (il+ cells). At three embryonic time points (E12.5, 14.5, and 16.5), no cell staining for both markers was observed. In fact, green and red fluorescent cells could be observed as positive / negative images of each other. Microdissected ker+ or il+ cells were collected in Gene Amp tubes (Applied Biosystems, Foster City, CA) filled with 75 μl of RNeasy lysis buffer (RLT; Qiagen, Hilden, Germany) containing 0.14 M β-mercaptoethanol and 200 ng of polyinosinic acid (Sigma).

[0060] RNA isolation, amplification, labeling and microarray hybridization Laser-captured samples were incubated at 42°C for 20 minutes and then chilled on ice. Samples were stored at -80°C until further processing. After thawing, an equal volume of 70% ethanol was added to each sample and then transferred to an RNeasy MinElute spin column (Qiagen). RNA was cleaned up according to the manufacturer's instructions, eluted with 14 μl of RNase-free water, and adjusted to 4 μl by vacuum drying. Two rounds of linear mRNA amplification were required to generate sufficient amounts of cRNA. Two-cycle cDNA synthesis and biotin-labeled cRNA synthesis were performed according to the GeneChip Eukaryotic Sample and Array Processing Manual (Affymetrix, Santa Clara, CA). The GeneChip Poly-A RNA Control Kit (Affymetrix) was used as a "spike-in" control. In vitro transcription of the second cDNA strand was performed in the first round of amplification using the MEGAscript T7 kit (Ambion, Austin, TX), yielding 112–457 ng of aRNA. Starting with 100 ng of aRNA from the first round, a second round of amplification was performed to yield 11–86 μg of cRNA using the GeneChip in vitro transcription (IVT) labeling kit. The labeled RNA was hybridized to Mouse Genome MG-430_2.0 GeneChip arrays (Affymetrix). Hybridization was performed with 12.5 μg of biotin-labeled RNA at 45°C for 16 hours under continuous rotation. Arrays were stained with streptavidin-phycoerythrin (SAPE) in an Affymetrix Fluidics station, followed by staining with an anti-streptavidin antibody and a secondary SAPE stain. The arrays were then scanned with an Agilent Laserscanner (Affymetrix).

[0061] statistical analysis Affymetrix probe-level data were analyzed using FARMS (Factor Analysis for Robust Microarray Summarization). 1 The raw intensity values were log2 transformed to obtain normally distributed data. First, to reduce the complexity of the high-dimensional data (n genes vs. p samples), we used Spectral Map Analysis, an unsupervised multivariate projection method. 2 Spectral Map Analysis provides unbiased identification of the main clusters of genes and subjects present in the dataset. Second, LIMMA (Linear Models for Microarray Data) was applied. 3 We investigated differential gene expression between two cell origins (ker+ cells vs. il+ cells) because this method uses information from the entire gene pool and is robust to experiments using a small number of arrays. 3 Third, again using LIMMA, we tested for differences in expression profiles across embryonic age between Vegf120 / 120 knockout and wild-type littermates through a two-way interaction between Vegf genotype and time. Data from E12.5 and E14.5 were pooled because we were only interested in differences in the temporal profiles between E16.5 and E14.5 and E12.5. This test was performed separately for ker+ and il+ samples because these two tissues were derived from the same embryo. Models such as LIMMA assume that all samples were collected randomly and independently. If ker+ and il+ were analyzed simultaneously, correcting for this dependency would have required overly complex models. Genomic variation from a single interaction of tissue type (ker+ samples vs. il+ samples) was also not accounted for by the LIMMA analysis. Assignment of differential expression along the whole genome was performed using MACT (Microarray Chromosome Analysis Tool).

[0062] Results / Discussion At birth, O2 and CO2 must be exchanged in the lungs across a wide interface of distal airways and blood vessels. Embryonic lung development in the mouse undergoes significant changes at E (= post-gestational day) 16.5. 1 At this point, the intertwining airway and vascular branches grow rapidly by proliferating and refining their distal branches. The distal airways or respiratory tubes grow by prenatally redividing into thin-walled sacs. These sacs eventually differentiate into alveoli after birth. 2 Thin-walled airways require flat cells to facilitate gas transport. Therefore, phenotypic differentiation into flat airway cells, which occurs around E16.5, is a critical stage in embryonic lung development. Epithelial cells lining the airways are derived from the branching foregut mesoderm. From E16.5, epithelial cells in the distal airways begin to flatten, while proximal cells do not lose their columnar shape. The most distal of these cells, lining the sacs and alveoli, develop a flat or even squamous morphology by E18.5. Capillaries are lined with flat endothelial cells and serve as the distal vessels of vascular branches. Endothelial cells lining the pulmonary vessels are derived from mesodermal mesenchyme. Their proliferation must closely coordinate with that of their epithelial counterparts to provide a wide alveolar-capillary interface for initiating gas exchange at birth. Reciprocal crosstalk between the endoderm-derived airway epithelium and the surrounding mesodermal mesenchyme is initiated during early lung morphogenesis. 3,4 Starting at E9.5, Fgf10, produced by mesenchymal cells in the surrounding mesoderm, is the most important signal for endoderm branching. Close interactions with at least Shh, Bmp, TGF-β, and Wnt signaling factors regulate this early branching mechanism. However, the molecular mechanisms underlying the subsequent cell phenotypic changes and epithelial-endothelial crosstalk at E16.5 remain poorly understood.

[0063] To gain further insight into late lung differentiation after E12.5, we developed an RNA-friendly immunohistochemical staining protocol for laser capture microdissection of epithelial cells in the developing airway. We reasoned that downstream gene expression profiling of RNA isolated from airway cells sharing common epithelial antigens at various embryonic ages would highlight their transcriptional changes over time. This program would at least reflect epithelial characteristics, preferably those of the lung airway type. The same hypothesis was tested for lung cells by selecting ubiquitously expressed endothelial markers at various embryonic ages. Furthermore, we incorporated this approach into a mouse knockout model with late abnormal lung branching morphogenesis. We chose the Vegf120 / 120 model because peripheral airway and vascular differentiation were significantly enhanced in these Vegf120 / 120 knockout embryonic lungs. 5,6,7 Wild-type epithelial and endothelial cells are expected to express a set of airway and vascular differentiation genes that are lacking in their Vegf120 / 120 knockout counterparts. Vegf120 / 120 mice lack VEGF-A isoforms 164 and 188, but still express isoform 120. VEGF-A isoforms 164 and 188 (Vegf 164 and Vegf 188 ) bind more tightly to the extracellular matrix than the more soluble VEGF120 variants and are concentrated locally around the distal airways. The standard view is that lung epithelial cells secrete these VEGF-A isoforms, thereby promoting VEGF production. 164 and Vegf 188 It has been shown that VEGF promotes local proliferation of pulmonary endothelial cells through stimulation of the receptor tyrosine kinases Flk1 (VEGF receptor-2) and Flt1 (VEGF receptor-1). The localized proliferation of endothelial cells refines the pulmonary vascular tree and allows for the coordinated proliferation of epithelial cells. 8,9This epithelial-endothelial crosstalk enables gas exchange at birth by forming tight junctions between the alveoli of the distal airways and the capillaries of the pulmonary vasculature. However, this type of interaction cannot explain the presence of VEGF-A in mesenchymal cells surrounding distal airway epithelial cells.

[0064] Immunohistochemical staining was performed on frozen tissue sections cut from the thorax of embryos isolated at E12.5, E14.5, and E16.5 (Figure 2). Genomic distributions for these embryos are shown in Table S1. We selected antibodies that bound to junctional epithelial or endothelial antigens and showed sufficient bandwidth within the embryonic timeframe of our study. For labeling of epithelial cells lining the airways (Ker+ cells), we chose anti-cytokeratin (against cytokeratins 4, 5, 6, 8, 10, 13, and 18) because primitive and differentiated epithelial cells systemically express various keratin intermediate filaments. Endothelial cells in the same tissue sections were also stained for early stage keratin expression in mice. 10 and late endothelial cells 11,12 The isolectin GS-IB4 (Griffonia simplicifolia), which binds to IL+ cells, was stained with Alexa-fluor-594 conjugate. Cell staining for both immunohistochemical markers was not observed at the three embryonic time points. Approximately 300–400 ker+ and IL+ cells were selectively isolated by laser capture microdissection. Two rounds of linear mRNA amplification yielded sufficient cRNA for hybridization to Affymetrix Mouse 430_2.0 Genechips.

[0065] First, we tested whether downstream gene expression profiling reflected differentiation with respect to fetal age and epithelial versus endothelial origin. Preliminary, unsupervised analysis of gene expression data revealed that gene expression changes during fetal development explained the largest amount of variation (35%) in the dataset. This variation is well illustrated in the first principal component (x-axis or PC1) of the spectral map. 13(Figure 3). Genes showing the strongest changes in expression across the three embryonic stages are located at the extremes of the x-axis. One of these genes, surfactant-related protein C (Sftpc), is known to exhibit significant physiological upregulation during embryonic development, and sufficient amounts of its protein product are required for normal respiration at birth. 14,15 The second principal component (y-axis or PC2), which explained another 17% of the variance in the dataset, could be attributed to differences in gene expression across cell origins. Some of the most extreme probe sets on the PC2 axis represent genes known to be highly characteristic of either the lung epithelium or endothelium. Among the eight most extreme probe sets, an illustrative endothelial gene was the CD93 antigen (CD93). 16 and claudin 5 (Cldn5) 17 , and on the other side of the y-axis, Forkhead box A1 (Foxa1) as a prominent epithelial gene. 18 and keratin 8 (Krt8) 19 We identified the following. Overlaying the various samples on the spectral map revealed their distribution along the first two major components. The ker+ and il+ groups were clearly separated according to their cellular origin along PC2 (Figure 3). ker+ samples clustered in the epithelial gene direction, while il+ samples clustered in the endothelial gene direction. At the same fetal age, both cellular origins clustered on PC1. This indicates that the overall developmental gene expression changes are similar for epithelial (ker+) and endothelial (il+) cells. When applying unsupervised data-driven analyses, samples clustered overall with respect to cellular origin (ker+ vs. il+ cells) and fetal age. Spectral map analysis highlights the superior resolution of gene expression profiles by selective laser capture microdissection. An independent supervised univariate (gene-by-gene) analysis of the effect of sample tissue origin (ker+ vs. il+) further confirmed that ker+ and il+ cells corresponded to epithelial or endothelial cells at the genomic level, respectively (Figure 1).

[0066] Next, we charted the transcriptional profiles associated with abnormal branching morphogenesis in the Vegf120 / 120 knockout phenotype in il+ and ker+ cells. We examined whether the expression profiles of all genes significantly differed between Vegf120 / 120 knockout and wild-type (Vegf+ / +) littermates across fetal ages. This difference in fetal age-dependent expression profiles between Vegf+ / + and Vegf120 / 120 expanded the genome roadmap in three directions. Several genes, such as Hmr, were identified that showed clear upregulation across fetal ages only in the Vegf+ / + genotype (Fig. 2). The Vegf120 / 120 genotype exhibited impaired fetal age-dependent induction.

[0067] First, we clarified the cause of the structural defects in the airways of Vegf120 / 120 knockout lungs. Wild-type Ker+ cells strongly expressed an extra 44 epithelial-specific genes at E16.5 compared with their Vegf120 / 120 knockout counterparts. Epidermal Differentiation Complex (EDC) genes dominated this expression profile (Fig. 4). Within this EDC subset, S100a8 and S100a9 were represented, which are VEGF-A responsive. 20 These genes are known chemoattractants. Other elements of EDCs, such as small proline-rich region (Sprr) genes and late components of the epithelial cornified envelope, were also present. Along with this EDC subset, the cytoskeletal keratins Krt2-6 were coexpressed in wild-type Ker+ cells at E16.5. Three genes from the serine-cysteine proteinase inhibitor and SCC (stratified epithelial cell-secreted protein gene) complex completed the cohort of upregulated genes (Figure 4).

[0068] Studies of the interaction between embryonic age and genotype in wild-type il+ cells again revealed a strong upregulation of a limited set of genes at E16.5. This response led to the adoption of an epithelial-specific transformation program in wild-type il+ cells at E16.5 compared with Vegf120 / 120 knockout cells. As in wild-type ker+ cells, the EDC cluster, SCC cluster, cysteine proteinase inhibitor, and exclusive keratin genes were clearly upregulated throughout embryonic age. Riken1110020A10, corresponding to the Dsc1 gene, was strongly upregulated in wild-type il+ cells at E16.5. Furthermore, Pkp1 (plakophilin 1) showed an identical transcriptional profile at E16.5 (Figure 5). A highly logical pattern appeared to drive the clustered upregulation of these genes. Keratins are intermediate filament proteins that provide structural strength to cells, most commonly epithelial cells. 21 Proteins encoded by the EDC and SCC clusters, as well as serine-cysteine proteinase inhibitors, reinforce this keratin network. Dsc1 (desmocollin 1) encodes one of the proteins that make up the desmosome. 22,23 Intermediate keratin filaments are linked to intercellular desmosomes, which together with gaps and adherens junctions form cell junctions. The protein encoded by Pkp1 is, among other things, a positive regulator of desmosomal protein content. 24,25, a component of the desmosome complex itself. Pkp1 also links intermediate keratin filaments to cadherin proteins at the zonula appressin membrane. These results reveal receptor tyrosine kinase stimulation by VEGF-A isoforms 164 and 188 as a master switch in the assembly of the desmosome / intermediate filament machinery in the lung. This machinery adds another building block to the cytoskeleton and cell-cell organization on top of the Wnt / β-catenin-dependent zonula appressin membrane (E-cadherin). Indeed, upregulation of Eps8l1 at E16.5 in wild-type il+ cells even revealed a direct inhibition of actin, a key structural protein with no analogue in the intermediate filament system. The coordinated and clustered expression of these cytoskeletal and desmosome genes enables the formation of flat or squamous cell structures in the distal airways.

[0069] Second, in addition to the activation of genes encoding specific structural proteins, an intriguing finding was the upregulation of Mapkapk3 in wild-type il+ cells at E16.5. Mapkapk3 mediates ERK and p38 signaling in stress and mitogen responses, e.g., in VEGF-A stimulation of endothelial cells. 26 Integrating pathways. Cdkn2b (p15ink4b or Ink4b), part of the Ink4b-ARF-Ink4a tumor suppressor locus, was simultaneously upregulated. Significant evidence indicates repression of this locus by the associated Polycomb group (Pcg) repressor complex. Mapkapk3 27 Dissociation of the Pcg complex by activation or overexpression of Pcg leads to derepression of this locus. This brake pedal on the cell cycle allows differentiation during growth stimuli. 28 Indeed, cell cycle arrest that allows epithelial transformation of IL+ cells is mediated by Vegf expression in the lung. 164 and Vegf 188Interestingly, the upregulation of Krt5, Krt14, and Tcfap2c strikingly resembles the expression fingerprint of basal cell progenitors in the airway epithelium. 29 The basal cell phenotype appears only at birth in lung airway epithelium and generally binds isolectins. On the other hand, expression of Krt1, EDC, and SCC cluster genes is associated with the squamous differentiation program. The proteins ΔNp63 and TAap63 drive the keratinocyte precursor and squamous differentiation programs, respectively. The Trp63 gene, encoding these two proteins, was upregulated in wild-type il+ cells. As mentioned above, staining of cells for both anti-cytokeratins (anti-4, 5, 6, 8, 10, 13, and 18) and the isolectin GS-IB4 was not observed at the time points studied. The lack of Krt1 and Krt14 binding by anti-cytokeratin antibodies therefore allowed the identification of a specific epithelial transformation program in wild-type il+ cells at E16.5. It is unlikely that ker+ epithelial cells undergo this epithelial transformation program. This would require the anti-cytokeratin antibodies to lose their binding ability to avoid ker+ labeling. At the same time, Ker+ cells would have to stain exclusively with the isolectin GS-IB4. Consequently, we propose that lung Il+ cells contain a reservoir of cells that develop into epithelial maturation at E16.5. In other words, lung mesenchymal Il+ cells contain cells with both endothelial and epithelial potential.

[0070] Third, the gene represented by Affymetrix probe 1437019_at (RIKEN cDNA 2200001I15 gene) and the gene represented by Affymetrix probe 1437145_s_at (RIKEN cDNA 2310002J15 gene) were both upregulated at E16.5 in wild-type anti-cytokeratin 4-5-6-8-10-13-18-stained epithelial cells and wild-type GS-IB4-binding cells. We found that these two genes (although lacking biological annotation) closely co-expressed with the squamous and basal cell transcriptional programs. They play important roles in the production and regeneration of differentiated airway cells (Figures 6 and 7, Tables 1 and 2). Starting from the longest contig constructed from the sequenced clones, we found human homologs of the RIKEN cDNA 2200001I15 gene transcript (Fig. 8) and the RIKEN cDNA 2310002J15 gene transcript (Fig. 10). Furthermore, protein sequence alignment confirmed the existence of human homologs of the translated proteins of the RIKEN cDNA 2200001I15 gene (Fig. 9) and the RIKEN cDNA 2310002J15 gene (Fig. 11). These genes and their protein products are involved in regenerative functions in the respiratory system ( r egenerative function in the r Because RIKEN is involved in the espiratory system, we identified R2R genes for the mammalian homologs of the RIKEN cDNA 2200001I15 gene, transcript, and protein. 1 The name R2R was added to the mammalian homologues of the RIKEN cDNA 2310002J15 gene, transcript, and protein. 2 The name is proposed.

[0071] Fourth, we found that VEGF-A was expressed in Ker+ and Il+ cells regardless of wild-type or Vegf120 / 120 knockout status. Furthermore, the gene encoding VEGF receptor 1 (Flk-1 or Kdr) was not only abundantly expressed in Il+ cells but also significantly increased in Ker+ cells at E14.5. This pattern of VEGF-A and VEGF receptor expression challenges the classical view of the mesenchyme passively waiting for VEGF-A stimulation from the lung epithelium. This is essentially consistent with recent studies demonstrating the requirement for endogenous VEGF-A expression and autocrine signaling for endothelial cell survival. 30,31 Genomic footprinting of wild-type il+ cells versus Vegf120 / 120 knockout il+ cells also revealed that il+ cells are involved in delivering epithelial transformation stimuli. Upregulation of Fgfbp1, Lgals7, Lgals3, and Il18 in wild-type il+ cells at E16.5 corresponded to these stimuli. Upregulation of the gene encoding fibroblast growth factor binding protein 1 (Fgfbp1) indicates that primitive FGF-regulated lung sprouting is also active during the final stages of lung differentiation. Fgfbp1 acts by concentrating FGF2 and is a sensitive regulator of epithelial proliferation and differentiation in response to FGF stimuli from the mesenchyme. In this regard, the FGF receptor 2 gene (Fgfr2) was abundantly expressed in both the ker+ and il+ compartments.

[0072] In summary, we performed selective laser capture microdissection of cells sharing specific markers at various embryonic ages. This enabled us to distinguish gene expression profiles related to embryonic age, cell origin, and Vegf genotype. The transcriptional programs revealed by this approach highlighted the importance of intermediate filaments and desmosomal networks in the refinement of lung architecture. This mechanism adds another building block to the cytoskeleton and cell-cell organization on top of the Wnt / β-catenin-dependent adherens junction (E-cadherin). Intermediate filaments provide the necessary strength for cells exposed to massive mechanical and oxidative stress. Not surprisingly, lung cells adopt the same defensive genomic program as skin cells exposed to the same insults. In parallel with the cytoskeletal complexity, a basal cell progenitor program is required by IL+ cells during late embryonic development, which is Vegf164-188 dependent.

[0073] We have shown that downregulation of the FAM25 family and C9orf169 gene expression reduces KRT14 expression. Furthermore, we have shown that downregulation of the FAM25 family (human R2R 1 homologue) and C9orf169 (human R2R 2 Homolog) expression is VEGFA / VEGF 165 These genes are known to be upregulated by VEGFA / VEGF. 165 How do they function in this pathway? To clarify the specific role of R2R homologs in this pathway, siRNA-mediated knockdown of R2R homologs can be used. Indeed, the role of R2R homologs is to regulate VEGFA / VEGF signaling in cells. 165 VEGFA and VEGF regulate the response 165 Basal expression of the isoform is high in the lung epithelium. The function and structure of the epithelium is much more complex than its endothelial counterpart. Therefore, typical VEGFA / VEGF isoforms that 'grow and proliferate' in the endothelium are not expressed in the lung epithelium. 165The effect needs to be further refined in the endothelium. How can this be achieved? Expression of R2R homologs leads to the simultaneous regulation of HIF1A signaling (conferring oxygen tolerance) and specific (PERP) anti-apoptotic pathways. This allows the (re)generation of robust epithelial cells (which have a major defense barrier against stress) without conferring unlimited proliferative capacity. In other words, regulation of specific anti-apoptotic pathways does not 'permit' general apoptosis resistance, which would lead to the dangerous situation of cell immortalization, which would develop into cancer cells.

[0074] This study sheds new light on certain human lung diseases. In preterm neonates, late embryonic lung development is impaired if the neonate is born prematurely. Insufficient levels of surfactant protein in the alveoli lead to severe respiratory distress in a large group of premature infants. Instillation of surfactant into the neonatal lung has helped prevent and treat respiratory failure in premature infants. However, the high oxygen concentration and tensile stress of mechanical ventilation still lead to chronic lung injury or bronchopulmonary dysplasia (BPD). Progressive squamous differentiation in the distal airways of premature infants can prevent this debilitating condition. The intermediate filament network and synchronized replenishing of the basal cell pool may be crucial in research for the cure of certain adult lung diseases. The adoption of a squamous phenotype, accompanied by the expression of several EDC cluster genes, is a hallmark of squamous metaplasia in the airways of adults with chronic obstructive pulmonary disease (COPD). However, this defense mechanism against noxious stimuli is accompanied by a decrease in regenerative basal cells. 32 In contrast, embryos successfully develop a basal cell reservoir with a clear roadmap while simultaneously expressing a squamous differentiation program. This roadmap serves as a guide for pharmacological intervention in the intermediate filament or basal cell transcriptional machinery. It also points to transformed IL+ cells as the origin of lung epithelial cells.

[0075] R2R 1 and R2R 2 Functional characteristics of In mouse embryos, the intermediate filament gene family VEGF-A (mouse isoform Vegf 164 = VEGF isoform 165 )-dependent expression of these intermediate filament genes was confirmed. Expression of these intermediate filament genes leads to differentiation of lung epithelial cells and development of the basal cell program in lung mesenchymal cells. Furthermore, the intermediate filament gene VEGF-A (mouse isoform Vegf 164 = VEGF isoform 165 )-dependent expression was also confirmed in adult human primary epithelial cells. 165 Stimulation of these cells with VEGF leads to upregulation of intermediate filament gene expression. 165 R2R is downregulated by specific siRNA. In summary, intermediate filament gene expression serves as a paradigm for airway differentiation and regeneration. 1 and R2R 2 plays a specific role in this expression program.

[0076] R2R 1 R2R 1 Expression of VEGF-A (murine isoform Vegf) in mice 164 = human isoform VEGF 165 ) dependent. Lung mesenchymal cells (GS-IB4 positive staining cells) acquire the basal epithelial cell gene expression program. 1 Expression of is essential for mesenchymal-epithelial transition (MET). R2R in MET 1 The role of R2R has now been confirmed in embryonic tissues rather than in the lung. Completion of the ventricular septum in the heart is achieved by MET. In mice, R2R 1 is strongly expressed in the developing ventricular septum and is expressed in the mouse isoform Vegf 164 = human isoform VEGF 165 ) dependency. Conversely, R2R1 Expression is absent in the developing right ventricular outflow tract, the development of which is known to be MET-independent (see Figure 12). R2R 1 is a candidate gene for mesenchymal-epithelial transition in mice and humans. Expression of this gene is regulated by MET, which regulates VEGF-A (the mouse isoform Vegf 164 = human isoform VEGF 165 ) effect. The reverse process of MET is EMT (epithelial-mesenchymal transition). EMT is an essential part of cancer progression and metastasis. Therefore, R2R 1 may play an important role in cancer biology and treatment.

[0077] Furthermore, in silico analysis has revealed that R2R 1 It turns out that the protein product of R2R appears to interact with the ribosome. 1 The interaction of protein structures with the ribosome has potential importance for drug development in the fields of cancer biology and MET.

[0078] R2R 2 R2R 2 was found to be highly expressed in adult human primary lung epithelial cells. 1 The expression of VEGF-A (VEGF isoform) was upregulated over time. 165 ) dependence was found. 2 The protein product is important for the normal differentiation and maintenance of the adult lung epithelium (see Figure 13).

Claims

1. A method for identifying or obtaining an agent that promotes expression of the R2R 1 and / or R2R 2 gene and is useful for suppressing expression of the BCL2A1 gene or promoting expression of the MAP2K4 gene, comprising: (i) contacting the R2R 1 and / or R2R 2 gene with a test agent and detecting modulation of R2R 1 and / or R2R 2 gene expression; and (ii) comparing the results obtained from step (i) with the results obtained from a control method in which the R2R 1 and / or R2R 2 genes are not contacted with the test agent; Including, If the expression level of the R2R 1 and / or R2R 2 genes obtained from step (i) is higher than the expression level of the R2R 1 and / or R2R 2 genes obtained from the control method, the test agent is useful for promoting the expression of the MAP2K4 gene or suppressing the expression of the BCL2A1 gene; The aforementioned method, wherein the R2R 1 and / or R2R 2 gene is encoded by a sequence selected from the group consisting of the sequences represented by SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10 and 11.

2. The method of claim 1, which is carried out in a cell-based or cell-free system that has been adjusted to contain the R2R 1 and / or R2R 2 genes.

3. The method of claim 2, further comprising transfecting the cells with a nucleic acid comprising the R2R 1 and / or R2R 2 genes.

4. The method described in claim 3, wherein the nucleic acid is a vector.

5. The method of claim 1, wherein the test agent is selected from the group consisting of nucleic acids, antisense oligonucleotides, proteins, peptides, amino acids, antibodies, carbohydrates, and small organic molecules.

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